Cyclin d polynucleotides, polypeptides and uses thereof

ABSTRACT

The invention provides isolated polynucleotides, specifically Cyclin D polynucleotides, and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content and/or composition of plants for the purpose of increasing transformation efficiency.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application No.60/101,551 filed Sep. 23, 1998, to U.S. patent application Ser. No.09/398,858 filed Sep. 20, 1999, now U.S. Pat. No. 6,518,487, U.S. patentapplication Ser. No. 10/320,230 filed Dec. 16, 2002, now abandoned, toU.S. patent application Ser. No. 10/348,110 filed Jan. 21, 2003, nowabandoned, and to U.S. patent application Ser. No. 11/560,550 filed Nov.16, 2006, all of which are herein incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acids and methods for modulatingtheir expression in plants.

BACKGROUND OF THE INVENTION

Cell division plays a crucial role during all phases of plantdevelopment. The continuation of organogenesis and growth responses to achanging environment requires precise spatial, temporal anddevelopmental regulation of cell division activity in meristems (and incells with the capability to form new meristems such as in lateral rootformation). Such control of cell division is also important in organsthemselves (i.e. separate from meristems per se), for example, in leafexpansion, secondary growth, and endoreduplication.

A complex network controls cell proliferation in eukaryotes. Regulatorypathways communicate environmental constraints, such as nutrientavailability, mitogenic signals such as growth factors or hormones, ordevelopmental cues such as the transition from vegetative toreproductive. Ultimately, these regulatory pathways control the timing,frequency (rate), plane and position of cell divisions.

The basic mechanism of cell cycle control is conserved among eukaryotes.A catalytic protein serine/threonine kinase and an activating cyclinsubunit control progress through the cell cycle. The protein kinase isgenerally referred to as a cyclin-dependent-kinase (CDK), whose activityis modulated by phosphorylation and dephosphorylation events and bytheir association with regulatory subunits called cyclins. CDKs requireassociation with cyclins for activation, and the timing of activation islargely dependent upon cyclin expression. CDKs are a family ofserine/threonine protein kinases that regulate individual cell cycletransitions.

Eukaryote genomes typically encode multiple cyclin and CDK genes. Inhigher eukaryotes, different members of the CDK family act in differentstages of the cell cycle. Cyclin genes are classified according to thetiming of their appearance during the cell cycle. In addition to cyclinand CDK subunits, CDKs are often physically associated with otherproteins that alter localization, substrate specificity, or activity. Afew examples of such CDK interacting proteins are the CDK inhibitors,members of the Retinoblastoma-associated protein (Rb) family, and theConstitutive Kinase Subunit (CKS).

The protein kinase activity of the complex is regulated by feedbackcontrol at certain checkpoints. At such checkpoints the CDK activitybecomes limiting for further progress. When the feedback control networksenses the completion of a checkpoint, CDK is activated and the cellpasses through to the next checkpoint. Changes in CDK activity areregulated at multiple levels, including reversible phosphorylation ofthe cell cycle factors, changes in subcellular localization of thecomplex, and the rates of synthesis and destruction of limitingcomponents. P. W. Doerner, Cell Cycle Regulation in Plants, PlantPhysiol. (1994) 106:823-827.

Plants have unique developmental features that distinguish them fromother eukaryotes. Plant cells do not migrate, and thus only celldivision, expansion and programmed cell death determine morphogenesis.Organs are formed throughout the entire life span of the plant fromspecialized regions called meristems. In addition, many differentiatedcells have the potential to both dedifferentiate and to reenter the cellcycle. There are also numerous examples of plant cell types that undergoendoreduplication, a process involving nuclear multiplication withoutcytokinesis. The study of plant cell cycle control genes is expected tocontribute to the understanding of these unique phenomena. O. Shaul etal., Regulation of Cell Division in Arabidopsis, Critical Reviews inPlant Sciences, 15(2):97-112 (1996).

There is evidence to suggest that cells must be dividing fortransformation to occur. It has also been observed that dividing cellsrepresent only a fraction of cells that transiently express a transgene.Furthermore, the presence of damaged DNA in non-plant systems (similarto DNA introduced by particle gun or other physical means) has been welldocumented to rapidly induce cell cycle arrest (W. Siede, Cell cyclearrest in response to DNA damage: lessons from yeast, Mutation Res.337(2):73-84). Therefore, to optimize transformation it would bedesirable to provide a method for increasing the number of cellsundergoing division.

Cell division in higher eukaryotes is controlled by two main checkpointsin the cell cycle that prevent the cell from entering either M- orS-phase of the cycle prematurely. Evidence from yeast and mammaliansystems has repeatedly shown that over-expression of key cell cycleactivating genes can either trigger cell division in non-dividing cells,or stimulate division in previously dividing cells (i.e. the duration ofthe cell cycle is decreased and cell size is reduced). Examples of geneswhose over-expression has been shown to stimulate cell division includecyclins (see, e.g. Doerner, P. et al., Nature (1996) 380:520-423; Wang,T. C., et al., Nature (1994) 369:669-671; Quelle D. E., et al., GenesDev. (1993) 7:1559-1571, E2F transcription factors (see, e.g. Johnson D.G. et al., Nature (1993) 365:349-352; Lukas, J. et al., (1996) Mol.Cell. Biol. 16:1047-1057), cdc25 (see, e.g. Bell, M. H. et al., (1993)Plant Molecular Biology 23:445-451; Draetta, D. et al., (1996) BBA1332:53-63), and mdm2 (see, e.g. Teoh, G. et al., (1997) Blood90:1982-1992). Conversely, other gene products have been found toparticipate in negative regulation and/or checkpoint control,effectively blocking or retarding progression through the cell cycle.(See MacLachlan, T. K. et al., (1995) Critical Rev. Eukaroytic GeneExpression 5(2):127-156).

Current methods for genetic engineering in maize require a specific celltype as the recipient of new DNA. These cells are found in relativelyundifferentiated, rapidly growing callus cells or on the scutellarsurface of the immature embryo (which gives rise to callus).Irrespective of the delivery method currently used, DNA is introducedinto literally thousands of cells, yet transformants are recovered atfrequencies of 10⁻⁵ relative to transiently-expressing cells.Exacerbating this problem, the trauma that accompanies DNA introductiondirects recipient cells into cell cycle arrest and accumulating evidencesuggests that many of these cells are directed into apoptosis orprogrammed cell death. (Bowen et al., Tucson International Mol. Biol.Meetings).

Over the period between 1950 and 1980, the increase in maize productionworldwide outpaced both wheat and rice. Despite a temporary downswing inthe early to mid-1980's (due to both environmental and politicalfactors) world maize production has risen steadily from around 145million tons in 1950 to nearly 500 million tons by 1990. Increases inyield and harvested area have been the predominant contributors toenhanced world production; with yield playing the major role inindustrialized countries and area expansion being most important indeveloping countries. Yet, over the next ten years it's also predictedthat meeting the demand for corn worldwide will require an additional20% over current production (Dowswell, C. R., Paliwal, R. L., Cantrell,R. P. (1996) Maize in the Third World, Westview Press, Boulder, Colo.).

The components most often associated with maize productivity are grainyield or whole-plant harvest for animal feed (in the forms of silage,fodder, or stover). Thus the relative growth of the vegetative orreproductive organs might be preferred, depending on the ultimate use ofthe crop. Whether the whole plant or the ear are harvested, overallyield will depend strongly on vigor and growth rate. In modern maizehybrids, the impact of heterosis on overall plant vigor and yield hasbeen unarguably demonstrated (Duvick, D. N. (1984) In: Geneticcontributions to yield gains in five major crop plants. W. R. Fehr, ed.CSSA, Madison, Wis.). Corn breeders since the 1930's have beenselectively breeding by identifying inbreds that in combination producehybrid vigor well beyond either parent. Surprisingly little is knownabout why hybrids are so much larger than their parent inbreds, althoughthere are some interesting observations in the literature. In metabolicstudies, heterosis (increases over either parent) has been observed forphysiological traits such as P uptake by roots (Baliger and Barber,1979; Nielsen and Barber, 1978), but for many enzymatic traits thehybrid is often intermediate to the inbred parents (Hageman, R. H.,Leng, E. R., Dudley, J. W. (1967) Adv. Agron. 19:45-86; Chevalier, P.,Schrader, L. E. (1977) Crop Sci. 17:897-901; Schrader, L. E. (1974) CropSci. 14:201-205; Schrader, L. E. (1985) pp 79-89. In: Exploitation ofphysiological and genetic variability to enhance crop productivity.Harper, J. E. ed. Am. Soc. Plant Physiol. Rockville, Md., Schrader, L.E., Cataldo, D. A., Peterson, D. M., Vogelzang, R. D. (1974) PlantPhysiol. 32:337-341).

Anatomical data is less confusing. In summarizing data from an earlierpublication, Kiesselbach states that approximately 10% of the increasedvigor of the hybrid over its inbred parents is due to cell enlargement,and 90% can be accounted for simply by increased cell numbers(Kiesselbach, T. A. 1922, 1949. The Structure and Reproduction of Corn,Nebraska Agric. Exp. Stn. Res. Bull. 161). This evidence for enhancedcell divisions contributing to increased maize vigor remainsunchallenged. Recently it was shown that overexpressing a B cyclin inArabidopsis resulted in increased root biomass and the root cells weresmaller (indicative of accelerated cell division), but the overall plantmorphology was not perturbed (Doerner et al., 1996). Similarly,expression of maize CycD genes in corn will enhance growth and biomassaccumulation.

Other more specialized applications exist for these genes at the wholeplant level. It has been demonstrated that endoreduplication occurs innumerous cell types within plants, but this is particularly prevalent inmaize endosperm, the primary seed storage tissue. Under the direction ofendosperm-specific promoters, expression of CycD genes (and possiblyexpression of CycD in conjunction with genes that inhibit mitosis) willfurther stimulate the process of endoreduplication.

SUMMARY OF THE INVENTION

Generally, it is the object of the present invention to provide nucleicacids and proteins relating to the control of cell division.

It is another object of the present invention to provide nucleic acidsand proteins that can be used to identify other interacting proteinsinvolved in cell cycle regulation.

It is another object of the present invention to provide antigenicfragments of the proteins of the present invention.

It is another object of the present invention to provide transgenicplants comprising the nucleic acids of the present invention.

It is another object of the present invention to provide methods formodulating, in a transgenic plant, the expression of the nucleic acidsof the present invention.

It is another object of the present invention to provide a method forincreasing the number of cells undergoing cell division.

It is another object of the present invention to provide a method forincreasing crop yield.

It is another object of the present invention to provide a method forimproving transformation frequencies.

It is another object of the present invention to provide a method forproviding a positive growth advantage in a plant comprising modulatingCycD protein expression.

It is another object of the present invention to provide a method formodulating cell growth.

It is another object of the present invention to provide a method formodulating cell division.

It is another object of the present invention to provide a method formodulating plant height or size.

It is another object of the present invention to provide a method forproviding a positive growth advantage.

It is another object of the present invention to provide a method forincreasing the growth rate.

It is another object of the present invention to provide a method forenhancing or inhibiting organ growth, for example seed, root, shoot,ear, tassel, stalk, pollen, stamen.

It is another object of the present invention to provide a method forproducing organ ablation.

It is another object of the present invention to provide a method forproducing parthenocarpic fruits.

It is another object of the present invention to provide a method forproducing male sterile plants.

It is another object of the present invention to provide a method forenhancing embryogenic response, i.e. size or growth rate.

It is another object of the present invention to provide a method forincreasing callus induction.

It is another object of the present invention to provide a method forpositive selection.

It is another object of the present invention to provide a method forincreasing plant regeneration.

It is another object of the present invention to provide a method foraltering the percent of time that cells are arrested, i.e. in G1 or G0stages of the cell cycle.

It is another object of the present invention to provide a method foraltering the amount of time a cell spends in a particular cell cycle.

It is another object of the present invention to provide a method forimproving in cells the response to environmental stress such as drought,heat, or cold.

It is another object of the present invention to provide a method forincreasing the number of pods per plant.

It is another object of the present invention to provide a method forincreasing the number of seeds per pod or ear.

It is another object of the present invention to provide a method foraltering the lag time in seed development.

It is another object of the present invention to provide a method forproviding hormone independent cell growth.

It is another object of the present invention to provide a method forincreasing growth rate of cells in bioreactors.

Therefore, in one aspect, the present invention relates to an isolatednucleic acid comprising a member selected from the group consisting of:

-   -   (a) a polynucleotide that encodes a polypeptide of SEQ ID NOS:        1, 11, 13, or 21;    -   (b) a polynucleotide amplified from a monocot nucleic acid        library using the primers of SEQ ID NOS: 3-10, 15-20 or 23-30;    -   (c) a polynucleotide having 20 contiguous bases of SEQ ID NOS:        1, 11, 13, or 21;    -   (d) a polynucleotide encoding a monocot cyclin D protein;    -   (e) a polynucleotide having at least 70% identity to the entire        coding region of SEQ ID NOS: 1, 11, 13, or 21, wherein the %        identity is determined by GCG/bestfit program using a gap        creation penalty of 50 and a gap extension penalty of 3;    -   (f) a polynucleotide that hybridizes under stringent conditions        to a nucleic acid characterized by SEQ ID NOS: 1, 11, 13, or 21,        wherein the conditions include a wash in 0.1×SSC at 60 to 65°        C.;    -   (g) a polynucleotide characterized by the sequences set forth in        SEQ ID NOS: 1, 11, 13, or 21;    -   (h) An isolated nucleic acid amplified from a Zea mays nucleic        acid library using the primers of SEQ ID NOS: 3-10, 15-20 or        23-30;    -   (i) a polynucleotide complementary to a polynucleotide of (a)        through (g); and    -   (j) a polynucleotide having the sequence of ATCC deposit having        the Designation No. 98847 or 98848.

In another aspect, the present invention relates to recombinantexpression cassettes, comprising the nucleic acid operably linked to apromoter.

In some embodiments, the nucleic acid is operably linked in antisenseorientation to the promoter.

In another aspect, the present invention is directed to a host celltransfected with the recombinant expression cassette as described,supra.

In a further aspect, the present invention relates to an isolatedprotein comprising a polypeptide of at least 10 contiguous amino acidsencoded by the isolated nucleic acid. In some embodiments, thepolypeptide has a sequence selected from the group consisting of SEQ IDNOS: 2, 12, 14, and 22.

In another aspect, the present invention relates to an isolated nucleicacid comprising a polynucleotide of at least 25 nucleotides in lengthwhich selectively hybridizes under stringent conditions to a nucleicacid selected from the group consisting of SEQ ID NOS: 1, 11, 13, and21, or a complement thereof. In some embodiments, the isolated nucleicacid is operably linked to a promoter.

In yet another aspect, the present invention relates to an isolatednucleic acid comprising a polynucleotide, the polynucleotide having atleast 80% sequence identity to an identical length of a nucleic acidselected from the group consisting of SEQ ID NOS: 1, 11, 13, and 21 or acomplement thereof.

In another aspect, the present invention relates to an isolated nucleicacid comprising a polynucleotide having a sequence of a nucleic acidamplified from a Zea mays nucleic acid library using the primersselected from the group consisting of SEQ ID NOS: 3-10, 15-20, and 23-30or complements thereof. In some embodiments, the nucleic acid library isa cDNA library.

In another aspect, the present invention relates to a recombinantexpression cassette comprising a nucleic acid amplified from a libraryas referred to supra, wherein the nucleic acid is operably linked to apromoter.

In some embodiments, the present invention relates to a host celltransfected with this recombinant expression cassette.

In some embodiments, the present invention relates to a protein of thepresent invention that is produced from this host cell.

In an additional aspect, the present invention is directed to anisolated nucleic acid comprising a polynucleotide encoding a polypeptidewherein: (a) the polypeptide comprises at least 10 contiguous amino acidresidues from a first polypeptide selected from the group consisting ofSEQ ID NOS: 2, 12, 14, and 22; (b) the polypeptide does not bind toantisera raised against the first polypeptide which has been fullyimmunosorbed with the first polypeptide; and (c) the polypeptide has amolecular weight in non-glycosylated form within 10% of the firstpolypeptide.

In a further aspect, the present invention relates to a heterologouspromoter operably linked to a non-isolated polynucleotide of the presentinvention, wherein the polypeptide is encoded by a nucleic acidamplified from a nucleic acid library.

In yet another aspect, the present invention relates to a transgenicplant comprising a recombinant expression cassette comprising a plantpromoter operably linked to any of the isolated nucleic acids of thepresent invention. The present invention also provides transgenic seedfrom the transgenic plant.

In a further aspect, the present invention relates to a method ofmodulating expression of the genes encoding the proteins of the presentinvention in a plant, comprising the steps of (a) transforming a plantcell with a recombinant expression cassette comprising a polynucleotideof the present invention operably linked to a promoter; (b) growing theplant cell under plant growing conditions; and (c) inducing expressionof the polynucleotide for a time sufficient to modulate expression ofthe genes in the plant. Expression of the genes encoding the proteins ofthe present invention can be increased or decreased relative to anon-transformed control plant.

-   -   In another aspect of the invention an isolated protein is        provided comprising a member selected from the group consisting        of:    -   (a) a polypeptide comprising at least 25 contiguous amino acids        of SEQ ID NOS: 2, 12, 14, or 22;    -   (b) a polypeptide which is a monocot cyclin D protein;    -   (c) a polypeptide comprising at least 65% sequence identity to        SEQ ID NOS: 2, 12, 14, or 22, wherein the % sequence identity is        based on the entire sequence and is determined by GAP 10 using        default parameters;    -   (d) a polypeptide encoded by a nucleic acid of claim 1; and    -   (e) a polypeptide characterized by SEQ ID NO: 2, 12, 14, or 22.

DEFINITIONS

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The terms defined below are more fullydefined by reference to the specification as a whole.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “antibody” includes reference to antigen binding forms ofantibodies (e.g., Fab, F(ab)₂). The term “antibody” frequently refers toa polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, which specifically bind andrecognize an analyte (antigen). However, while various antibodyfragments can be defined in terms of the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments such as single chain Fv, chimeric antibodies (i.e.,comprising constant and variable regions from different species),humanized antibodies (i.e., comprising a complementarity determiningregion (CDR) from a non-human source) and heteroconjugate antibodies(e.g., bispecific antibodies).

The term “antigen” includes reference to a substance to which anantibody can be generated and/or to which the antibody is specificallyimmunoreactive. The specific immunoreactive sites within the antigen areknown as epitopes or antigenic determinants. These epitopes can be alinear array of monomers in a polymeric composition—such as amino acidsin a protein—or consist of or comprise a more complex secondary ortertiary structure. Those of skill will recognize that all immunogens(i.e., substance capable of eliciting an immune response) are antigens;however some antigens, such as haptens, are not immunogens but may bemade immunogenic by coupling to a carrier molecule. An antibodyimmunologically reactive with a particular antigen can be generated invivo or by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors. See, e.g., Huse etal., Science 246:1275-1281 (1989); and Ward, et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996).

As used herein, “antisense orientation” includes reference to a duplexpolynucleotide sequence that is operably linked to a promoter in anorientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

As used herein, “chromosomal region” includes reference to a length ofchromosome that can be measured by reference to the linear segment ofDNA that it comprises. The chromosomal region can be defined byreference to two unique DNA sequences, i.e., markers.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide of the present invention isimplicit in each described polypeptide sequence and incorporated hereinby reference.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservativelymodified variants typically provide similar biological activity as theunmodified polypeptide sequence from which they are derived. Forexample, substrate specificity, enzyme activity, or ligand/receptorbinding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe native protein for it's native substrate. Conservative substitutiontables providing functionally similar amino acids are well known in theart.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Serine (S), Threonine (T);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).    See also, Creighton (1984) Proteins W.H. Freeman and Company.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolum (Proc. Natl. Acad.Sci., U.S.A. 82:2306-2309 (1985)), or the ciliate Macronucleus, may beused when the nucleic acid is expressed using these organisms.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al., Nucl. Acids Res. 17:477-498(1989)). Thus, the maize preferred codon for a particular amino acid canbe derived from known gene sequences from maize. Maize codon usage for28 genes from maize plants are listed in Table 4 of Murray et al.,supra.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of, a native (non-synthetic), endogenous, catalytically activeform of the specified protein. A full-length sequence can be determinedby size comparison relative to a control that is a native(non-synthetic) endogenous cellular form of the specified nucleic acidor protein. Methods to determine whether a sequence is full-length arewell known in the art including such exemplary techniques as northern orwestern blots, primer extension, 51 protection, and ribonucleaseprotection. See, e.g., Plant Molecular Biology: A Laboratory Manual,Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to knownfull-length homologous (orthologous and/or paralogous) sequences canalso be used to identify full-length sequences of the present invention.Additionally, consensus sequences typically present at the 5′ and 3′untranslated regions of mRNA aid in the identification of apolynucleotide as full-length. For example, the consensus sequenceANNNNAUGG, where the underlined codon represents the N-terminalmethionine, aids in determining whether the polynucleotide has acomplete 5′ end. Consensus sequences at the 3′ end, such aspolyadenylation sequences, aid in determining whether the polynucleotidehas a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell that contains a vector and supports thereplication and/or expression of the expression vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledenous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

By “immunologically reactive conditions” or “immunoreactive conditions”is meant conditions which allow an antibody, generated to a particularepitope, to bind to that epitope to a detectably greater degree (e.g.,at least 2-fold over background) than the antibody binds tosubstantially all other epitopes in a reaction mixture comprising theparticular epitope. Immunologically reactive conditions are dependentupon the format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment; or (2)if the material is in its natural environment, the material has beensynthetically (non-naturally) altered by deliberate human interventionto a composition and/or placed at a locus in the cell (e.g., genome orsubcellular organelle) not native to a material found in thatenvironment. The alteration to yield the synthetic material can beperformed on the material within or removed from its natural state. Forexample, a naturally occurring nucleic acid becomes an isolated nucleicacid if it is altered, or if it is transcribed from DNA that has beenaltered, by non-natural, synthetic (i.e., “man-made”) methods performedwithin the cell from which it originates. See, e.g., Compounds andMethods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S.Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in EukaryoticCells; Zarling et al., PCT/US93/03868. Likewise, a naturally occurringnucleic acid (e.g., a promoter) becomes isolated if it is introduced bynon-naturally occurring means to a locus of the genome not native tothat nucleic acid. Nucleic acids that are “isolated” as defined herein,are also referred to as “heterologous” nucleic acids.

Unless otherwise stated, the term “cell cycle nucleic acid” means anucleic acid comprising a polynucleotide (“cell cycle polynucleotide”)encoding a cell cycle polypeptide. A “cell cycle gene” refers to anon-heterologous genomic form of a full-length cell cyclepolynucleotide.

As used herein, “localized within the chromosomal region defined by andincluding” with respect to particular markers includes reference to acontiguous length of a chromosome delimited by and including the statedmarkers.

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A“polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes in that pair to be followed. A genotype may be defined byuse of one or a plurality of markers.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules that comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989);and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds.Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994 Supplement).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants which can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants. A particularly preferred plant is Zea mays.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof, thathave the essential nature of a natural ribonucleotide in that theyhybridize to nucleic acids in a manner similar to naturally occurringnucleotides. A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. Exemplary modifications aredescribed in most basic texts, such as, Proteins—Structure and MolecularProperties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993). Many detailed reviews are available on this subject, suchas, for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pp. 1-12 in PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) andRattan et al., Protein Synthesis: Posttranslational Modifications andAging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, asis well known and as noted above, that polypeptides are not alwaysentirely linear. For instance, polypeptides may be branched as a resultof ubiquitination, and they may be circular, with or without branching,generally as a result of posttranslation events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. Modifications can occur anywherein a polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. In fact, blockage of theamino or carboxyl group in a polypeptide, or both, by a covalentmodification, is common in naturally occurring and syntheticpolypeptides and such modifications may be present in polypeptides ofthe present invention, as well. For instance, the amino terminal residueof polypeptides made in E. coli or other cells, prior to proteolyticprocessing, almost invariably will be N-formylmethionine. Duringpost-translational modification of the peptide, a methionine residue atthe NH₂-terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells such Agrobacterium or Rhizobium.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue preferred”. Promoters thatinitiate transcription only in certain tissue are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” promoter is a promoter that is underenvironmental control. Examples of environmental conditions that mayeffect transcription by inducible promoters include anaerobic conditionsor the presence of light. Tissue specific, tissue preferred, cell typespecific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterthat is active under most environmental conditions.

The term “cell cycle polypeptide” refers to one or more amino acidsequences, in glycosylated or non-glycosylated form, involved in theregulation of cell division. The term is also inclusive of fragments,variants, homologs, alleles or precursors (e.g., preproproteins orproproteins) thereof. A “cell cycle protein” comprises a cell cyclepolypeptide.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The term “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogs of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, preferably 90% sequenceidentity, and most preferably 100% sequence identity (i.e.,complementary) with each other.

The term “specifically reactive”, includes reference to a bindingreaction between an antibody and a protein having an epitope recognizedby the antigen binding site of the antibody. This binding reaction isdeterminative of the presence of a protein having the recognized epitopeamongst the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to an analyte having the recognized epitope to asubstantially greater degree (e.g., at least 2-fold over background)than to substantially all other analytes lacking the epitope which arepresent in the sample.

Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, antibodies raised to the polypeptides of the presentinvention can be selected from those antibodies that are specificallyreactive with polypeptides of the present invention. The proteins usedas immunogens can be in native conformation or denatured so as toprovide a linear epitope.

A variety of immunoassay formats may be used to select antibodiesspecifically reactive with a particular protein (or other analyte). Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that can be used to determine selective reactivity.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Generallyhybridization is conducted for a time in the range of from four tosixteen hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with ≧90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceand its complement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution) it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, 100, or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide sequence a gap penalty is typically introduced andis subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65(1992), and Pearson, et al., Methods in Molecular Biology 24:307-331(1994). The BLAST family of programs which can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences that may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie andStates, Comput. Chem., 17:191-201 (1993)) low-complexity filters can beemployed alone or in combination.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g. chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions have “sequence similarity” or “similarity”.Means for making this adjustment are well known to those of skill in theart. Typically this involves scoring a conservative substitution as apartial rather than a full mismatch, thereby increasing the percentagesequence identity. Thus, for example, where an identical amino acid isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. The scoring of conservative substitutions is calculated, e.g.,according to the algorithm of Meyers and Miller, Computer Applic. Biol.Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. For purposes of defining the invention, % identity onthe nucleic acid level is determined by the BESTFIT DNA SequenceAlignment software on Genescape using a gap weight of 50 and a lengthweight of 3. For purposes of defining the invention, % identity on theamino acid level is determined by the BESTFIT DNA Sequence Alignmentsoftware on Genescape using a gap weight of 12 and a length weight of 4.

(e) (i) The term “substantial identity” of polynucleotide sequencesmeans that a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters. Oneof skill will recognize that these values can be appropriately adjustedto determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like. Substantial identityof amino acid sequences for these purposes normally means sequenceidentity of at least 85%, more preferably at least 90%, and mostpreferably at least 98%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.However, nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This mayoccur, e.g., when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

(e) (ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Optimal alignment can beconducted using the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970). An indication that two peptidesequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides which are “substantially similar” share sequencesas noted above except that residue positions which are not identical maydiffer by conservative amino acid changes.

By “two-hybrid system” is meant a screening method to identifyprotein-protein interactions, using a known gene (and its encodedproduct) as a “bait” or target and screening a library of expressedgenes and their corresponding encoded products for specific interactionswith the “bait” molecule. Methods for library construction and use ofvisual marker genes for yeast two-hybrid screens are well known in theart, and can be found in Sambrook, et al., 1990, Ausubel et al., 1990and G. Hannon and P. Bartel, Identification of interacting proteinsusing the two-hybrid system. Methods Mol. Cellular Biol. 5:289-297(1995).

DETAILED DESCRIPTION OF THE INVENTION

The CycD genes in plants encode proteins ranging from 37 to 44 kD. Thisprotein is necessary for progression from G1 into S-phase. The encodedprotein binds to CDK4, and this active cyclin D-CDK4 kinasehyperphosphorylates Rb, releasing the E2F transcription factor whichactivates DNA synthesis. G1/S phase cyclins were first isolated in yeast(Hadwiger et al., 1989; Richardson et al., 1989), and a few years laterin humans (Matsushime et al., 1991). Subsequently, it has been cloned invarious other organisms including plants. Three CycD isoforms have beenfound in both animals and plants, which are analogous to, and cancomplement function of, the three CLN genes originally identified inyeast. In mammalian cells, cyclins appear to be important integrators ofgrowth signals for cell cycle control. In plants, this aspect has beenbest characterized in Arabidopsis, with AtCycD2 and AtCycD3 expressionbeing induced by sucrose and cytokinin, respectively (Francis et al.,1998). AtCycD3 can also been induced by nitrate levels (Fuerst et al.,1998). CycD1 has been cloned in Arabidopsis thaliana (Soni et al., 1995;EMBL accession number X83369), Antirrhinum majus and Helianthustuberosum. Cyclin D2 has been cloned in Arabidopsis (Soni et al, 1995;X83370), and CycD3 has been cloned in Arabidopsis (Soni et al., 1995;X83371), Antirrhinum, Helianthus (Freeman and Muray, unpublished),Nicotiana and Medicago (Dahl et al., 1995; X88864). No monocot homologshave been reported. In the present invention, we describe the fulllength clone of the maize CycD gene (designated ZmCycD).

In addition to the positive influence of transient cell cyclestimulation, stable expression of positive cell cycle regulators wouldbe a benefit for positive selection schemes in the recovery oftransgenic plants and plant cells. In a population of cells and/orcallus growing in vitro, cells expressing a gene such as CycD will havea differential growth advantage based simply on their accelerateddivision rate. It would be expected that these transgenic cells orcell/clusters would grow more rapidly than their non-transformedcounterparts in culture, permitting ready identification oftransformants. Such a positive growth advantage (imparted by expressionof a gene such as CycD, or CycD plus another cell cycle component),would also be beneficial in other types of transformation strategies,including as examples, protoplast transformation, leaf basetransformation and transformation of cells in meristems. Such growthstimulation may also extend transformation protocols to tissues normallyno amenable to culture. Examples would include such tissues as portionsof leaves (in which the cells do not normally divide), scutellum fromrecalcitrant inbreds (in which cells typically are not induced to dividein culture), and nodal tissues, etc.

Of particular interest is the use of cell cycle genes such as CycD toimpart a positive growth advantage to cells in the meristem, includingapical initials. The apical initials in angiosperm shoot meristems aredefined by their position within the meristem. If an apical initial cellbecomes compromised relative to neighboring cells in the meristem, itwill be replaced by an adjacent neighbor that is not at a disadvantage.This new cell assumes the role of the apical initial. Conversely,transgenic cells adjacent to the apical initials with a positive growthadvantage can, over time (i.e. through successive cell generations),out-compete the wild-type apical initials, eventually replacing thesecells and establishing a homogeneous transformed meristem.

There can also be organ and/or whole plant impacts to such cell cycletransgene expression.

REFERENCES

Renaudin, J-P., Doonan, J. H., Freeman, D., Hashimoto, J., Hirt, H.,Inze, D., Jacobs, T., Kouchi, H., Rouze, P., Sauter, M., Savoure, A.,Sorrell, D. A., Sundaresan, V., and Murray, J. A. H. 1996. Plantcyclins: a unified nomenclature for plant A-, B- and D-type cyclinsbased on sequence organization. Plant Molecular Biology 32:1003-1018.

Dahl, M., Meskiene, I., Boegre, L., Ha, D. T. C., Swoboda, I., Hubmann,R., Hirt, H. and Heberle-Bors, E. 1995. The D-type alfalfa cyclin genecycMs4 complements G 1 cyclin-deficient yeast and is induced in the G-1phase of the cell cycle. Plant Cell 7(11):1847-1857.

Murray, J. A. H., Freeman, D., Greenwood, J., Huntley, R., Makkerk, J.Riou-Khamlichi, C., Sorrell, D. A., Cockcroft, C., Carmichael., J. P.,Soni, R. and Shah, Z. H. 1998. Plant D cyclins and retinoblastomaprotein homologues. In: Plant Cell Division, (Francis, D., Dudits, D.and Inze D., eds.), Portland Press, London.

Fuerst, R. A. U. A., Soni, R., Murray, J. A. H. and Lindsey, K. 1998.Modulation of cyclin transcript levels in cultured cells of Arabidopsisthaliana. Plant Physiol. 112:1023-1033.

Hadwiger, J. A., Wittenberg, C., Richardson, H. E., de Barros Lopes, M.and Reed, S. I. 1989. A family of cyclin homologs that control the G1phase in yeast. Proc. Natl. Acad. Sci. USA 86(16):6255-6259.

Matsushime, H., Roussel, M. F. and Sherr, C. J. 1991. Novel mammaliancyclins (CYL genes) expressed during G1. Cold Spring Harb. Symp. Quant.Biol. 56:69-74.

Richardson, H. E., Wittenberg, C., Cross, F and Reed, S. I. 1989. Anessential G1 function for cyclin-like proteins in yeast. Cell59(6):1127-1133.

Soni, R., Carmichael, J. P., shah, Z. H. and Murray, J. A. H. 1995. Afamily of cyclin D homologs from plants differentially controlled bygrowth regulators and containing the conserved retinoblastoma proteininteraction motif. Plant Cell 7:85-103.

The present invention provides, inter alia, compositions and methods formodulating (i.e., increasing or decreasing) the total levels of proteinsof the present invention and/or altering their ratios in plants. Thus,the present invention provides utility in such exemplary applications asthe regulation of cell division. The polypeptides of the presentinvention can be expressed at times or in quantities that are notcharacteristic of non-recombinant plants.

In particular, modulating cell cycle proteins is expected to provide apositive growth advantage and increase crop yield. Cell cycle nucleicacids can be adducted to a second nucleic acid sequence encoding aDNA-binding domain, for use in two-hybrid systems to identifyCycD-interacting proteins. It is expected that modulating the level ofcell cycle protein, i.e. over-expression, will increaseendoreduplication which is expected to increase the size of the seed,the size of the endosperm and amount of protein in the seed. The cellcycle protein can be used to affinity purify active maturation promotingfactor (MPF) or its components.

The present invention also provides isolated nucleic acid comprisingpolynucleotides of sufficient length and complementarity to a cell cyclegene to use as probes or amplification primers in the detection,quantitation, or isolation of gene transcripts. For example, isolatednucleic acids of the present invention can be used as probes indetecting deficiencies in the level of mRNA in screenings for desiredtransgenic plants, for detecting mutations in the gene (e.g.,substitutions, deletions, or additions), for monitoring upregulation ofexpression or changes in enzyme activity in screening assays ofcompounds, for detection of any number of allelic variants(polymorphisms) of the gene, or for use as molecular markers in plantbreeding programs. The isolated nucleic acids of the present inventioncan also be used for recombinant expression of cell cycle polypeptides,or for use as immunogens in the preparation and/or screening ofantibodies. The isolated nucleic acids of the present invention can alsobe employed for use in sense or antisense suppression of one or morecell cycle genes in a host cell, tissue, or plant. Attachment ofchemical agents that bind, intercalate, cleave and/or crosslink to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation. Further, using a primer specificto an insertion sequence (e.g., transposon) and a primer whichspecifically hybridizes to an isolated nucleic acid of the presentinvention, one can use nucleic acid amplification to identity insertionsequence inactivated cell cycle genes from a cDNA library prepared frominsertion sequence mutagenized plants. Progeny seed from the plantscomprising the desired inactivated gene can be grown to a plant to studythe phenotypic changes characteristic of that inactivation. See, Toolsto Determine the Function of Genes, 1995 Proceedings of the FiftiethAnnual Corn and Sorghum Industry Research Conference, American SeedTrade Association, Washington, D.C., 1995. Additionally, non-translated5′ or 3′ regions of the polynucleotides of the present invention can beused to modulate turnover of heterologous mRNAs and/or proteinsynthesis. Further, the codon preference characteristic of thepolynucleotides of the present invention can be employed in heterologoussequences, or altered in homologous or heterologous sequences, tomodulate translational level and/or rates.

The present invention also provides isolated proteins comprisingpolypeptides including an amino acid sequence from the cell cyclepolypeptides (e.g., preproenzyme, proenzyme, or enzymes) as disclosedherein. The present invention also provides proteins comprising at leastone epitope from a cell cycle polypeptide. The proteins of the presentinvention can be employed in assays for enzyme agonists or antagonistsof enzyme function, or for use as immunogens or antigens to obtainantibodies specifically immunoreactive with a protein of the presentinvention. Such antibodies can be used in assays for expression levels,for identifying and/or isolating nucleic acids of the present inventionfrom expression libraries, or for purification of cell cyclepolypeptides.

The isolated nucleic acids of the present invention can be used over abroad range of plant types, including species from the genera Cucurbita,Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium,Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum,Sorghum, Picea, and Populus. Preferred plants include corn, soybeans,sorghum, sunflower, wheat, rice, alfalfa and canola.

Nucleic Acids

The present invention provides, inter alfa, isolated nucleic acids ofRNA, DNA, and analogs and/or chimeras thereof, comprising a cell cyclepolynucleotide.

A. Polynucleotides Encoding A Protein of SEQ ID NOS: 2, 12, 14, or 22 orConservatively Modified or Polymorphic Variants Thereof

The present invention provides isolated heterologous nucleic acidscomprising a cell cycle polynucleotide, wherein the polynucleotideencodes a cell cycle polypeptide, disclosed herein in SEQ ID NOS: 2, 12,14, or 22, or conservatively modified or polymorphic variants thereof.Those of skill in the art will recognize that the degeneracy of thegenetic code allows for a plurality of polynucleotides to encode for theidentical amino acid sequence. Such “silent variations” can be used, forexample, to selectively hybridize and detect allelic variants ofpolynucleotides of the present invention. Accordingly, the presentinvention includes polynucleotides of SEQ ID NOS: 1, 11, 13, or 21, andsilent variations of polynucleotides encoding a polypeptide of SEQ IDNOS: 2, 12, 14, or 22. The present invention further provides isolatednucleic acids comprising polynucleotides encoding conservativelymodified variants of a polypeptide of SEQ ID NOS: 2, 12, 14, or 22.Conservatively modified variants can be used to generate or selectantibodies immunoreactive to the non-variant polypeptide.

B. Polynucleotides Amplified from a Zea mays Nucleic Acid Library

As indicated in (b), supra, the present invention provides isolatednucleic acids comprising cell cycle polynucleotides, wherein thepolynucleotides are amplified from a Zea mays nucleic acid library. Zeamays lines B73, PHRE1, A632, BMS-P2#10, W23, and Mo17 are known andpublicly available. Other publicly known and available maize lines canbe obtained from the Maize Genetics Cooperation (Urbana, Ill.).

The nucleic acid library may be a cDNA library, a genomic library, or alibrary generally constructed from nuclear transcripts at any stage ofintron processing. Generally, a cDNA nucleic acid library will beconstructed to comprise a majority of full-length cDNAs. Often, cDNAlibraries will be normalized to increase the representation ofrelatively rare cDNAs.

Total RNA Isolation: Libraries can be made from a variety of maizetissues but for optimal results one should isolate RNA's frommitotically active tissues such as shoot meristems, shoot meristemcultures, callus and suspension cultures, immature ears and tassels, andyoung seedlings. Since cell cycle proteins are typically expressed atspecific cell cycle stages it may be possible to enrich for such raremessages using exemplary cell cycle inhibitors such as aphidicolin,hydroxyurea, mimosine, and double-phosphate starvation methods to blockcells at the G1/S boundary. Cells can also be blocked at this stageusing the double phosphate starvation method. Hormone treatments thatstimulate cell division, for example cytokinin, would also increaseexpression of the cell cycle RNA.

Full length cDNA libraries from such rapidly-dividing tissues (or cellsat the G1/S boundary) would provide opportunities for identifying fulllength, cell cycle related cDNAs. Full length cDNA libraries can beconstructed using the “Biotinylated CAP Trapper” method (Carninci, P.,et al., Genomics 37:327-336, 1996) or the “mRNA Cap Retention Procedure”(Edery, I., et al., Molecular and Cellular Biology 15:3363-3371, 1995).Full length cDNA libraries can be normalized to provide a higherprobability of sampling genes that express at low levels. Examples ofcDNA library normalization methods are summarized by Bento Soares(Bonaldo, M. F., et al., Genome Research 6:791-806, 1996).

Functional fragments of cell cycle protein can be identified using avariety of techniques such as restriction analysis, Southern analysis,primer extension analysis, and DNA sequence analysis. Function can alsobe determined by complementing yeast strains known to be mutant for G1cell cycle proteins with maize homologs. Primer extension analysis or S1nuclease protection analysis, for example, can be used to localize theputative start site of transcription of the cloned gene. Ausubel atpages 4.8.1 to 4.8.5; Walmsley et al., “Quantitative and QualitativeAnalysis of Exogenous Gene Expression by the 51 Nuclease ProtectionAssay,” in Methods in Molecular Biology, Vol. 7, Gene Transfer andExpression.

The general approach of such functional analysis involves subcloning DNAfragments of a genomic clone, cDNA clone or synthesized gene sequenceinto an expression vector, introducing the expression vector into aheterologous host, and relying on an assay system such as BrdUincorporation to monitor DNA synthesis in conjunction with variouswell-established visual methods to follow cell division (e.g. see T.Motomura, Cell cycle analysis in a multinucleate green alga, Boergensiaforbesti (Syphonoclades, Chlorophyta). Phycological Res. 44(1):11-17,and J. L. Kennard et al., Pre-mitotic nuclear migration in subsidiarymother cells of Tradescantia occurs in the G1 of the cell cycle. CellMotility and the Cytoskeleton 36:55-67). Methods for generatingfragments of a cDNA or genomic clone are well known. In addition,variants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like. See, for example, Ausubel,pages 8.0.3-8.5.9. Also, see generally, McPherson (ed.), DirectedMutagenesis: A Practical Approach, (IRL Press, 1991). Thus, the presentinvention also encompasses DNA molecules comprising nucleotide sequencesthat have substantial sequence similarity with SEQ ID NO: 1, 11, 13, or22 and encode CycD.

The polynucleotides of the present invention include those amplifiedusing the following primer pairs:

Primer Sets for ZmCycDa-1

1) Primer Sets Flanking ZmCycDa-1 cDNA:

Set #1: SEQ ID NO: 3 For01 5′ GCAAGCATGGTGCCGGGCTATGACTGC 3′SEQ ID NO: 4 Rev01 5′ AGCGGTGAGGAGCACACCTGAAGCGTACCA 3′ Set #2:SEQ ID NO: 3 For01 5′ GCAAGCATGGTGCCGGGCTATGACTGC 3′ SEQ ID NO: 5Rev02 5′ TCTATTCCTCTGCCGACCCCCATCCTT 3′ Set #3: SEQ ID NO: 6 For02 5′CCCCTCTCCACTTGAGAAGAACACAATTAG 3′ SEQ ID NO: 4 Rev01 5′AGCGGTGAGGAGCACACCTGAAGCGTACCA 3′ Set #4: SEQ ID NO: 6 For02 5′CCCCTCTCCACTTGAGAAGAACACAATTAG 3′ SEQ ID NO: 5 Rev02 5′TCTATTCCTCTGCCGACCCCCATCCTT 3′2) Primer Sets Inside ZmCycDa-1 cDNA:

Set #1: SEQ ID NO: 7 For01 5′ CGGGCTATGACTGCGCCGCCTCCGT 3′ SEQ ID NO: 8Rev01 5′ CTCCTCTTGCTTGTGGAAGAACTATGG 3′ Set #2: SEQ ID NO: 9 For02: 5′ATGGTGCCGGGCTATGACTGCGCCG 3′ SEQ ID NO: 10 Rev02: 5′TTAGAGTAGACGTCTAGTGATCCTT 3′

Primer Sets for ZmCycDb-1: 1) Primer Sets Inside ZmCycDb-1:

Set #1 SEQ ID NO: 15 For01: 5′ CACGCGCACCAGCCCACCGCCCAG 3′ SEQ ID NO: 16Rev01: 5′ TCCCATCGGATCTCCTCTAGCGCCC 3′

Primer Sets for ZmCycDc-1:

1) Primer Sets Flanking ZmCycDc-1 cDNA:

Set #1: SEQ ID NO: 23 For01: CAGTACCCCCACGCTGCACAG SEQ ID NO: 24Rev01: TCACGCTTGTTCTGTCGTCTTTACAC Set #2: SEQ ID NO: 25For02: GCTGCTGCAAGTCCGCAACCACTG SEQ ID NO: 26Rev02: CGCTTGTTCTGTCGTCTTTACACTG2) Primer Sets Inside ZmCycDc-1 cDNA:

Set #1: SEQ ID NO: 27 For01: 5′ ACCTCCATCCTCATCTGCCTGGAAGACSEQ ID NO: 28 Rev01: 5′ CTGGACTGCACTGCACTGCAATGC Set #2: SEQ ID NO: 29For02: 5′ CATCCTCATCTGCCTGGAAGACGGC SEQ ID NO: 30 Rev02: 5′AATGCACTGCCAGCAGCTGAGCT

The present invention also provides subsequences of full-length nucleicacids. Any number of subsequences can be obtained by reference to SEQ IDNOS: 1, 11, 13, or 21, and using primers which selectively amplify,under stringent conditions to: at least two sites to the polynucleotidesof the present invention, or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention, or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. A variety of methods for obtaining5′ and/or 3′ ends is well known in the art. See, e.g., RACE (RapidAmplification of Complementary Ends) as described in Frohman, M. A., inPCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H.Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc., SanDiego, 1990), pp. 28-38.); see also, U.S. Pat. No. 5,470,722, andCurrent Protocols in Molecular Biology, Unit 15.6, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995). Thus,the present invention provides cell cycle polynucleotides having thesequence of the cell cycle gene, nuclear transcript, cDNA, orcomplementary sequences and/or subsequences thereof.

Primer sequences can be obtained by reference to a contiguoussubsequence of a polynucleotide of the present invention. Primers arechosen to selectively hybridize, under PCR amplification conditions, toa polynucleotide of the present invention in an amplification mixturecomprising a genomic and/or cDNA library from the same species.Generally, the primers are complementary to a subsequence of theamplicon they yield. In some embodiments, the primers will beconstructed to anneal at their 5′ terminal end's to the codon encodingthe carboxy or amino terminal amino acid residue (or the complementsthereof) of the polynucleotides of the present invention. The primerlength in nucleotides is selected from the group of integers consistingof from at least 15 to 50. Thus, the primers can be at least 15, 18, 20,25, 30, 40, or 50 nucleotides in length. A non-annealing sequence at the5′end of the primer (a “tail”) can be added, for example, to introduce acloning site at the terminal ends of the amplicon.

The amplification primers may optionally be elongated in the 3′direction with additional contiguous nucleotides from the polynucleotidesequences, such as SEQ ID NOS: 1, 11, 13, or 21, from which they arederived. The number of nucleotides by which the primers can be elongatedis selected from the group of integers consisting of from at least 1 to25. Thus, for example, the primers can be elongated with an additional1, 5, 10, or 15 nucleotides. Those of skill will recognize that alengthened primer sequence can be employed to increase specificity ofbinding (i.e., annealing) to a target sequence.

The amplification products can be translated using expression systemswell known to those of skill in the art and as discussed, infra. Theresulting translation products can be confirmed as polypeptides of thepresent invention by, for example, assaying for the appropriatecatalytic activity (e.g., specific activity and/or substratespecificity), or verifying the presence of one or more linear epitopesthat are specific to a polypeptide of the present invention. Methods forprotein synthesis from PCR derived templates are known in the art andavailable commercially. See, e.g., Amersham Life Sciences, Inc., Catalog'97, p. 354.

C. Polynucleotides which Selectively Hybridize to a Polynucleotide of(A) or (B)

As indicated in (c), supra, the present invention provides isolatednucleic acids comprising cell cycle polynucleotides, wherein thepolynucleotides selectively hybridize, under selective hybridizationconditions, to a polynucleotide of paragraphs (A) or (B) as discussed,supra. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising thepolynucleotides of (A) or (B). For example, polynucleotides of thepresent invention can be used to identify, isolate, or amplify partialor full-length clones in a deposited library. In some embodiments, thepolynucleotides are genomic or cDNA sequences isolated from a Zea maysnucleic acid library. Preferably, the cDNA library comprises at least80% full-length sequences, preferably at least 85% or 90% full-lengthsequences, and more preferably at least 95% full-length sequences. ThecDNA libraries can be normalized to increase the representation of raresequences. Low stringency hybridization conditions are typically, butnot exclusively, employed with sequences having a reduced sequenceidentity relative to complementary sequences. Moderate and highstringency conditions can optionally be employed for sequences ofgreater identity. Low stringency conditions allow selectivehybridization of sequences having about 70% sequence identity and can beemployed to identify orthologous or paralogous sequences.

D. Polynucleotides Having at Least 60% Sequence Identity with thePolynucleotides of (A), (B) or (C)

As indicated in (d), supra, the present invention provides isolatednucleic acids comprising cell cycle polynucleotides, wherein thepolynucleotides have a specified identity at the nucleotide level to apolynucleotide as disclosed above in paragraphs (A), (B), or (C). Thepercentage of identity to a reference sequence is at least 60% and,rounded upwards to the nearest integer, can be expressed as an integerselected from the group of integers consisting of from 60 to 99. Thus,for example, the percentage of identity to a reference sequence can beat least 70%, 75%, 80%, 85%, 90%, or 95%.

Optionally, the polynucleotides of this embodiment will share an epitopewith a polypeptide encoded by the polynucleotides of (A), (B), or (C).Thus, these polynucleotides encode a first polypeptide that elicitsproduction of antisera comprising antibodies that are specificallyreactive to a second polypeptide encoded by a polynucleotide of (A),(B), or (C). However, the first polypeptide does not bind to antiseraraised against itself when the antisera has been fully immunosorbed withthe first polypeptide. Hence, the polynucleotides of this embodiment canbe used to generate antibodies for use in, for example, the screening ofexpression libraries for nucleic acids comprising polynucleotides of(A), (B), or (C), or for purification of, or in immunoassays for,polypeptides encoded by the polynucleotides of (A), (B), or (C). Thepolynucleotides of this embodiment embrace nucleic acid sequences thatcan be employed for selective hybridization to a polynucleotide encodinga polypeptide of the present invention.

Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure. Antibody screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 15amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT patent publication Nos.91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent publication Nos. 92/05258,92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754; and5,643,768. Peptide display libraries, vectors, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.).

E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and is Cross-Reactive to the Prototype Polypeptide

As indicated in (e), supra, the present invention provides isolatednucleic acids comprising cell cycle polynucleotides, wherein thepolynucleotides encode a protein having a subsequence of contiguousamino acids from a prototype cell cycle polypeptide. Exemplary prototypecell cycle polypeptides are provided in SEQ ID NOS. 2, 12, 14, or 23.The length of contiguous amino acids from the prototype polypeptide isselected from the group of integers consisting of from at least 10 tothe number of amino acids within the prototype sequence. Thus, forexample, the polynucleotide can encode a polypeptide having asubsequence having at least 10, 15, 20, 25, 30, 35, 40, 45, or 50,contiguous amino acids from the prototype polypeptide. Further, thenumber of such subsequences encoded by a polynucleotide of the instantembodiment can be any integer selected from the group consisting of from1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by anyinteger of nucleotides from 1 to the number of nucleotides in thesequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such as,but not limited to, a polypeptide encoded by the polynucleotide of (b),supra, or exemplary polypeptides of SEQ ID NOS. 2, 12, 14, or 23.Generally, however, a protein encoded by a polynucleotide of thisembodiment does not bind to antisera raised against the prototypepolypeptide when the antisera have been fully immunosorbed with theprototype polypeptide. Methods of making and assaying for antibodybinding specificity/affinity are well known in the art. Exemplaryimmunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

In a preferred assay method, fully immunosorbed and pooled antisera thatis elicited to the prototype polypeptide can be used in a competitivebinding assay to test the protein. The concentration of the prototypepolypeptide required to inhibit 50% of the binding of the antisera tothe prototype polypeptide is determined. If the amount of the proteinrequired to inhibit binding is less than twice the amount of theprototype protein, then the protein is said to specifically bind to theantisera elicited to the immunogen. Accordingly, the proteins of thepresent invention embrace allelic variants, conservatively modifiedvariants, and minor recombinant modifications to a prototypepolypeptide.

A polynucleotide of the present invention optionally encodes a proteinhaving a molecular weight as the non-glycosylated protein within 20% ofthe molecular weight of the full-length non-glycosylated cell cyclepolypeptides as disclosed herein. Molecular weight can be readilydetermined by SDS-PAGE under reducing conditions. Preferably, themolecular weight is within 15% of a full-length cell cycle polypeptide,more preferably within 10% or 5%, and most preferably within 3%, 2%, or1% of a full-length cell cycle polypeptide of the present invention.Molecular weight determination of a protein can be convenientlyperformed by SDS-PAGE under denaturing conditions.

Optionally, the polynucleotides of this embodiment will encode a proteinhaving a specific activity at least 20%, 30%, 40%, or 50% of the native,endogenous (i.e., non-isolated), full-length cell cycle polypeptide.Further, the proteins encoded by polynucleotides of this embodiment willoptionally have a substantially similar apparent dissociation constant(K_(m))) and/or catalytic activity (i.e., the microscopic rate constant,k_(cat)) as the native endogenous, full-length cell cycle protein. Thoseof skill in the art will recognize that k_(cat)/K_(m) value determinesthe specificity for competing substrates and is often referred to as thespecificity constant. Proteins of this embodiment can have ak_(cat)/K_(m) value at least 10% of the non-isolated full-length cellcycle polypeptide as determined using the substrate of that polypeptidefrom the cell cycle specific pathways, supra. Optionally, thek_(cat)/K_(m) value will be at least 20%, 30%, 40%, 50%, and mostpreferably at least 60%, 70%, 80%, 90%, or 95% the k_(cat)/K_(m) valueof the non-isolated, full-length cell cycle polypeptide. Determinationof k_(cat), K_(m) , and k_(cat)/K_(m) can be determined by any number ofmeans well known to those of skill in the art. For example, the initialrates (i.e., the first 5% or less of the reaction) can be determinedusing rapid mixing and sampling techniques (e.g., continuous-flow,stopped-flow, or rapid quenching techniques), flash photolysis, orrelaxation methods (e.g., temperature jumps) in conjunction with suchexemplary methods of measuring as spectrophotometry, spectrofluorimetry,nuclear magnetic resonance, or radioactive procedures. Kinetic valuesare conveniently obtained using a Lineweaver-Burk or Eadie-Hofstee plot.

F. Polynucleotides Complementary to the Polynucleotides of (A)-(E)

As indicated in (f), supra, the present invention provides isolatednucleic acids comprising cell cycle polynucleotides, wherein thepolynucleotides are complementary to the polynucleotides of paragraphsA-E, above. As those of skill in the art will recognize, complementarysequences base-pair throughout the entirety of their length with thepolynucleotides of (A)-(E) (i.e., have 100% sequence identity over theirentire length). Complementary bases associate through hydrogen bondingin double stranded nucleic acids. For example, the following base pairsare complementary: guanine and cytosine; adenine and thymine; andadenine and uracil.

G. Polynucleotides that are Subsequences of the Polynucleotides of(A)-(F)

As indicated in (g), supra, the present invention provides isolatednucleic acids comprising cell cycle polynucleotides, wherein thepolynucleotide comprises at least 15 contiguous bases from thepolynucleotides of (A) through (F) as discussed above. The length of thepolynucleotide is given as an integer selected from the group consistingof from at least 15 to the length of the nucleic acid sequence fromwhich the polynucleotide is a subsequence of. Thus, for example,polynucleotides of the present invention are inclusive ofpolynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75, or100 contiguous nucleotides in length from the polynucleotides of(A)-(F). Optionally, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4, or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100, or 200 nucleotides.

The subsequences of the present invention can comprise structuralcharacteristics of the sequence from which it is derived. Alternatively,the subsequences can lack certain structural characteristics of thelarger sequence from which it is derived. For example, a subsequencefrom a polynucleotide encoding a polypeptide having at least one linearepitope in common with a prototype sequence, may encode an epitope incommon with the prototype sequence. Alternatively, the subsequence maynot encode an epitope in common with the prototype sequence but can beused to isolate the larger sequence by, for example, nucleic acidhybridization with the sequence from which it's derived. Subsequencescan be used to modulate or detect gene expression by introducing intothe subsequence compounds that bind, intercalate, cleave and/orcrosslink to nucleic acids. Exemplary compounds include acridine,psoralen, phenanthroline, naphthoquinone, daunomycin orchloroethylaminoaryl conjugates.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot. In preferred embodiments the monocot is Zea mays.Particularly preferred is the use of Zea mays tissue from tassel andvegetative meristem.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the polynucleotide sequence—isgenerally a vector, adapter, or linker for cloning and/or expression ofa polynucleotide of the present invention. Use of cloning vectors,expression vectors, adapters, and linkers is well known in the art.Exemplary nucleic acids include such vectors as: M13, lambda ZAPExpress, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV,pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4, pWE15,SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/−, pSG5, pBK, pCR-Script, pET,pSPUTK, p3′SS, pOPRSVI CAT, pOPl3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406,pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambda MOSElox. Fora description of various nucleic acids see, for example, StratageneCloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and,Amersham Life Sciences, Inc., Catalog '97 (Arlington Heights, Ill.).

A. Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. While isolation ofRNA and construction of cDNA and genomic libraries is well known tothose of ordinary skill in the art, the following highlights some of themethods employed.

A1. mRNA Isolation and Purification

Total RNA from plant cells comprises such nucleic acids as mitochondrialRNA, chloroplastic RNA, rRNA, tRNA, hnRNA and mRNA. Total RNApreparation typically involves lysis of cells and removal of proteins,followed by precipitation of nucleic acids. Extraction of total RNA fromplant cells can be accomplished by a variety of means. Frequently,extraction buffers include a strong detergent such as SDS and an organicdenaturant such as guanidinium isothiocyanate, guanidine hydrochlorideor phenol. Following total RNA isolation, poly(A)⁺ mRNA is typicallypurified from the remainder RNA using oligo(dT) cellulose. Exemplarytotal RNA and mRNA isolation protocols are described in Plant MolecularBiology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin(1997); and, Current Protocols in Molecular Biology, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995). TotalRNA and mRNA isolation kits are commercially available from vendors suchas Stratagene (La Jolla, Calif.), Clonetech (Palo Alto, Calif.),Pharmacia (Piscataway, N.J.), and 5′-3′ (Paoli, Pa.). See also, U.S.Pat. Nos. 5,614,391; and, 5,459,253. The mRNA can be fractionated intopopulations with size ranges of about 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 kb.The cDNA synthesized for each of these fractions can be size selected tothe same size range as its mRNA prior to vector insertion. This methodhelps eliminate truncated cDNA formed by incompletely reversetranscribed mRNA.

A2. Construction of a cDNA Library

Construction of a cDNA library generally entails five steps. First,first strand cDNA synthesis is initiated from a poly(A)⁺ mRNA templateusing a poly(dT) primer or random hexanucleotides. Second, the resultantRNA-DNA hybrid is converted into double stranded cDNA, typically by acombination of RNAse H and DNA polymerase I (or Klenow fragment). Third,the termini of the double stranded cDNA are ligated to adaptors.Ligation of the adaptors will produce cohesive ends for cloning. Fourth,size selection of the double stranded cDNA eliminates excess adaptorsand primer fragments, and eliminates partial cDNA molecules due todegradation of mRNAs or the failure of reverse transcriptase tosynthesize complete first strands. Fifth, the cDNAs are ligated intocloning vectors and packaged. cDNA synthesis protocols are well known tothe skilled artisan and are described in such standard references as:Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997); and, Current Protocols in MolecularBiology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995). cDNA synthesis kits are availablefrom a variety of commercial vendors such as: Stratagene, and Pharmacia.

A number of cDNA synthesis protocols have been described which providesubstantially pure full-length cDNA libraries. Substantially purefull-length cDNA libraries are constructed to comprise at least 90%, andmore preferably at least 93% or 95% full-length inserts amongst clonescontaining inserts. The length of insert in such libraries can be from 0to 8, 9, 10, 11, 12, 13, or more kilobase pairs. Vectors to accommodateinserts of these sizes are known in the art and available commercially.See, e.g., the Stratagene lambda ZAP Express (cDNA cloning vector with 0to 12 kb cloning capacity).

An exemplary method of constructing a greater than 95% pure full-lengthcDNA library is described by Carninci et al., Genomics 37:327-336(1996). In that protocol, the cap-structure of eukaryotic mRNA ischemically labeled with biotin. By using streptavidin-coated magneticbeads, only the full-length first-strand cDNA/mRNA hybrids areselectively recovered after RNase I treatment. The method provides ahigh yield library with an unbiased representation of the starting mRNApopulation. Other methods for producing full-length libraries are knownin the art. See, e.g., Edery et al., Mol. Cell Biol. 15(6):3363-3371(1995); and, PCT Application WO 96/34981.

A3. Normalized or Subtracted cDNA Libraries

A non-normalized cDNA library represents the mRNA population of thetissue it was made from. Since unique clones are out-numbered by clonesderived from highly expressed genes their isolation can be laborious.Normalization of a cDNA library is the process of creating a library inwhich each clone is more equally represented.

A number of approaches to normalize cDNA libraries are known in the art.One approach is based on hybridization to genomic DNA. The frequency ofeach hybridized cDNA in the resulting normalized library would beproportional to that of each corresponding gene in the genomic DNA.Another approach is based on kinetics. If cDNA reannealing followssecond-order kinetics, rarer species anneal less rapidly and theremaining single-stranded fraction of cDNA becomes progressively morenormalized during the course of the hybridization. Specific loss of anyspecies of cDNA, regardless of its abundance, does not occur at any Cotvalue. Construction of normalized libraries is described in Ko, Nucl.Acids. Res., 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl.Acad. U.S.A. 88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685, and5,637,685. In an exemplary method described by Soares et al.,normalization resulted in reduction of the abundance of clones from arange of four orders of magnitude to a narrow range of only 1 order ofmagnitude, Proc. Natl. Acad. Sci. USA 91:9228-9232 (1994).

Subtracted cDNA libraries are another means to increase the proportionof less abundant cDNA species. In this procedure, cDNA prepared from onepool of mRNA is depleted of sequences present in a second pool of mRNAby hybridization. The cDNA:mRNA hybrids are removed and the remainingun-hybridized cDNA pool is enriched for sequences unique to that pool.See, Foote et al. in, Plant Molecular Biology: A Laboratory Manual,Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique,3(2):58-63 (1991); Sive and St. John, Nucl. Acids Res., 16(22):10937(1988); Current Protocols in Molecular Biology, Ausubel, et al., Eds.,Greene Publishing and Wiley-Interscience, New York (1995); and, Swaroopet al., Nucl. Acids Res., 19(8):1954 (1991). cDNA subtraction kits arecommercially available. See, e.g., PCR-Select (Clontech).

A4. Construction of a Genomic Library

To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation, e.g. using restriction endonucleases,and are ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends, andsequencing methods to verify the sequence of nucleic acids are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning, and screening methodologies arefound in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Vols. 1-3 (1989), Methods inEnzymology, Vol. 152, Guide to Molecular Cloning Techniques, Berger andKimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocolsin Molecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

A5. Nucleic Acid Screening and Isolation Methods

The cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled bytemperature, ionic strength, pH and the presence of a partiallydenaturing solvent such as formamide. For example, the stringency ofhybridization is conveniently varied by changing the polarity of thereactant solution through manipulation of the concentration of formamidewithin the range of 0% to 50%. The degree of complementarity (sequenceidentity) required for detectable binding will vary in accordance withthe stringency of the hybridization medium and/or wash medium. Thedegree of complementarity will optimally be 100 percent; however, itshould be understood that minor sequence variations in the probes andprimers may be compensated for by reducing the stringency of thehybridization and/or wash medium.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to clone flanking genomicsequences, 5′ untranslated regions and 3′ sequences, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes. Examples oftechniques sufficient to direct persons of skill through in vitroamplification methods are found in Berger, Sambrook, and Ausubel, aswell as Mullis et al., U.S. Pat. No. 4,683,202 (1987); and, PCRProtocols A Guide to Methods and Applications, Innis et al., Eds.,Academic Press Inc., San Diego, Calif. (1990). Commercially availablekits for genomic PCR amplification are known in the art. See, e.g.,Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

PCR-based screening methods have also been described. Wilfinger et al.describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.BioTechniques, 22(3): 481-486 (1997). In that method, a primer pair issynthesized with one primer annealing to the 5′ end of the sense strandof the desired cDNA and the other primer to the vector. Clones arepooled to allow large-scale screening. By this procedure, the longestpossible clone is identified amongst candidate clones. Further, the PCRproduct is used solely as a diagnostic for the presence of the desiredcDNA and does not utilize the PCR product itself. Such methods areparticularly effective in combination with a full-length cDNAconstruction methodology, supra.

B. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90-99 (1979); thephosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979);the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862 (1981); the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers, Tetra. Letts. 22(20):1859-1862(1981), e.g., using an automated synthesizer, e.g., as described inNeedham-VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984);and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis generally produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polynucleotide of the present invention,for example a cDNA or a genomic sequence encoding a full lengthpolypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

Cell cycle vectors were constructed using standard molecular biologytechniques. See, for example, Sambrook et al. (eds.) Molecular Cloning:a Laboratory Manual, Second Edition, (Cold Spring Harbor LaboratoryPress, cold Spring Harbor, N.Y. 1989). Plasmids are based on pUC18. Thevectors used in these experiments contain combinations of the same basicregulatory elements. The Omega prime (O′) 5-prine sequence is describedby Gallie et al., Nucl. Acids Res. 15:3257-3273 (1987). The selectivemarker gene, bar (Thompson et al., EMBO J. 6:2519-2523 (1987)), was usedin conjunction with bialaphos selection to recover transformants. TheCauliflower Mosaic Virus 35S promoter with a duplicated enhancer regionis described by Gardner et al., Nucl. Acid Res. 9:2871-2888 (1981). The79 bp Tobacco Mosaic Virus leader is described by Gallie et al., Nucl.Acid Res. 15:3257-3273 (1987) and was inserted downstream of thepromoter followed by the first intron of the maize alcohol dehydrogenasegene ADH1-S. Described by Dennis et al., Nucl. Acid Res. 12:3983-3990(1984). The 3′ sequence pinII is described by An et al., Plant Cell1:115-122 (1989).

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter,the GRP1-8 promoter, and other transcription initiation regions fromvarious plant genes known to those of skill.

Promoters A. Inducible Promoters

An inducible promoter can be operably linked to a nucleotide sequenceencoding ZmCycD. Optionally, the inducible promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a nucleotide sequence encoding ZmCycD. With an induciblepromoter the rate of transcription increases in response to an inducingagent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude that from the ACE1 system which responds to copper (Mett et al.,PNAS 90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen.Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen.Genet. 227:229-237 (1991). A particularly preferred inducible promoteris a promoter that responds to an inducing agent to which plants do notnormally respond. An exemplary inducible promoter is the induciblepromoter from a steroid hormone gene the transcriptional activity ofwhich is induced by a glucocorticosteroid hormone. Schena et al., Proc.Natl. Acad. Sci. U.S.A. 88:10421 (1991).

The expression vector comprises an inducible promoter operably linked toa nucleotide sequence encoding ZmCycD. The expression vector isintroduced into plant cells and presumptively transformed cells areexposed to an inducer of the inducible promoter. The cells can bescreened for the presence of ZmCycD protein by northern, RPA, or RT-PCR(using transgene specific probes/oligo pairs) BrdU or cell divisionassays, as described above.

B. Tissue-Specific or Tissue Preferred Promoters

A tissue-specific promoter can be operably linked to a nucleotidesequence encoding a ZmCycD protein. Optionally, the tissue-specificpromoter is operably linked to a nucleotide sequence encoding a signalsequence which is operably linked to a nucleotide sequence encodingZmCycD. Plants transformed with a gene encoding ZmCycD operably linkedto a tissue-specific promoter produce the ZmCycD protein exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include a seed-preferred promoter such as that from thephaseolin gene (Murai et al., Science 23:476-482 (1983) andSengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320-3324(1985)), napin promoter, β-conglycinin promoter soybean lectin promoter,maize 15 kD zein promoter, 22 kD zein promoter, γ-zein promoter, waxypromoter, shrunken 1 promoter, globulin 1 promoter and shrunken 2promoter (Thompson, et al.; BioEssays; Vol. 10; p. 108; (1989); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko etal., Nature 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell et al., Mol. Gen. Genet. 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero et al., Mol.Gen. Genet. 224:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)).

The expression vector comprises a tissue-specific or tissue-preferredpromoter operably linked to a nucleotide sequence encoding cell cycleprotein. The expression vector is introduced into plant cells. The cellsare screened for the presence of cell cycle protein by either BrdU orcell division assays, as described above.

C. Constitutive Promoters

A constitutive promoter can be operably linked to a nucleotide sequenceencoding a cell cycle protein or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a nucleotide sequence encoding cell cycle protein.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include the promoters fromplant viruses such as the 35S promoter from CaMV (Odell et al., Nature313:810-812 (1985)), Commelina yellow mottled virus (R. Torbert et al.,Plant Cell Rep. 17:284-287 (1988)) and the promoters from such genes asrice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730(1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genet.231:276-285 (1992) and Atanassova et al., Plant Journal 2(3):291-300(1992)).

The ALS promoter, a XbaI/NcoI fragment 5-prime to the Brassica napusALS3 structural gene (or a nucleotide sequence that has substantialsequence similarity to the XbaI/NcoI fragment), represents aparticularly useful constitutive promoter. Co-pending Pioneer Hi-BredInternational U.S. patent application Ser. No. 08/409,297.

The expression vector comprises a constitutive promoter operably linkedto a nucleotide sequence encoding cell cycle protein. The expressionvector is introduced into plant cells and presumptively transformedCELLS are screened for the presence of cell cycle protein by either BrdUor cell division assays, as described above.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds, or flowers. The operation of a promotermay also vary depending on its location in the genome. Thus, aninducible promoter may become fully or partially constitutive in certainlocations.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in recombinantexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter cell cycle content and/or composition in adesired tissue. Thus, in some embodiments, the nucleic acid constructwill comprise a promoter functional in a plant cell, such as in Zeamays, operably linked to a polynucleotide of the present invention.Promoters useful in these embodiments include the endogenous promotersdriving expression of a polypeptide of the present invention.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868), or isolated promoters can be introduced into a plantcell in the proper orientation and distance from a cell cycle gene so asto control the expression of the gene. Gene expression can be modulatedunder conditions suitable for plant growth so as to alter cell cyclecontent and/or composition. Thus, the present invention providescompositions, and methods for making, heterologous promoters and/orenhancers operably linked to a native, endogenous (i.e.,non-heterologous) form of a polynucleotide of the present invention.

Methods for identifying promoters with a particular expression pattern,in terms of, e.g., tissue type, cell type, stage of development, and/orenvironmental conditions, are well known in the art. See, e.g., TheMaize Handbook, Chapters 114-115, Freeling and Walbot, Eds., Springer,New York (1994); Corn and Corn Improvement, 3^(rd) edition, Chapter 6,Sprague and Dudley, Eds., American Society of Agronomy, Madison, Wis.(1988). A typical step in promoter isolation methods is identificationof gene products that are expressed with some degree of specificity inthe target tissue. Amongst the range of methodologies are: differentialhybridization to cDNA libraries; subtractive hybridization; differentialdisplay; differential 2-D gel electrophoresis; DNA probe arrays; andisolation of proteins known to be expressed with some specificity in thetarget tissue. Such methods are well known to those of skill in the art.Commercially available products for identifying promoters are known inthe art such as the Clontech (Palo Alto, Calif.) Universal GenomeWalkerKit.

For the protein-based methods, it is helpful to obtain the amino acidsequence for at least a portion of the identified protein, and then touse the protein sequence as the basis for preparing a nucleic acid thatcan be used as a probe to identify either genomic DNA directly, orpreferably, to identify a cDNA clone from a library prepared from thetarget tissue. Once such a cDNA clone has been identified, that sequencecan be used to identify the sequence at the 5′ end of the transcript ofthe indicated gene. For differential hybridization, subtractivehybridization and differential display, the nucleic acid sequenceidentified as enriched in the target tissue is used to identify thesequence at the 5′ end of the transcript of the indicated gene. Oncesuch sequences are identified, starting either from protein sequences ornucleic acid sequences, any of these sequences identified as being fromthe gene transcript can be used to screen a genomic library preparedfrom the target organism. Methods for identifying and confirming thetranscriptional start site are well known in the art.

In the process of isolating promoters expressed under particularenvironmental conditions or stresses, or in specific tissues, or atparticular developmental stages, a number of genes are identified thatare expressed under the desired circumstances, in the desired tissue, orat the desired stage. Further analysis will reveal expression of eachparticular gene in one or more other tissues of the plant. One canidentify a promoter with activity in the desired tissue or condition butthat do not have activity in any other common tissue.

To identify the promoter sequence, the 5′ portions of the clonesdescribed here are analyzed for sequences characteristic of promotersequences. For instance, promoter sequence elements include the TATA boxconsensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10bp located approximately 20 to 40 base pairs upstream of thetranscription start site. Identification of the TATA box is well knownin the art. For example, one way to predict the location of this elementis to identify the transcription start site using standard RNA-mappingtechniques such as primer extension, S1 analysis, and/or RNaseprotection. To confirm the presence of the AT-rich sequence, astructure-function analysis can be performed involving mutagenesis ofthe putative region and quantification of the mutation's effect onexpression of a linked downstream reporter gene. See, e.g., The MaizeHandbook, Chapter 114, Freeling and Walbot, Eds., Springer, New York(1994).

In plants, further upstream from the TATA box, at positions −80 to −100,there is typically a promoter element (i.e., the CAAT box) with a seriesof adenines surrounding the trinucleotide G (or T) N G. J. Messing etal., in Genetic Engineering in Plants, Kosage, Meredith and Hollaender,Eds., pp. 221-227 (1983). In maize, there is no well conserved CAAT boxbut there are several short, conserved protein-binding motifs upstreamof the TATA box. These include motifs for the trans-acting transcriptionfactors involved in light regulation, anaerobic induction, hormonalregulation, or anthocyanin biosynthesis, as appropriate for each gene.

Once promoter and/or gene sequences are known, a region of suitable sizeis selected from the genomic DNA that is 5′ to the transcriptionalstart, or the translational start site, and such sequences are thenlinked to a coding sequence. If the transcriptional start site is usedas the point of fusion, any of a number of possible 5′ untranslatedregions can be used in between the transcriptional start site and thepartial coding sequence. If the translational start site at the 3′ endof the specific promoter is used, then it is linked directly to themethionine start codon of a coding sequence.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. CellBiol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987).Such intron enhancement of gene expression is typically greatest whenplaced near the 5′ end of the transcription unit. Use of maize intronsAdh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. Seegenerally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds.,Springer, New York (1994).

The vector comprising the sequences from a polynucleotide of the presentinvention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, the nptII gene encodesresistance to the antibiotics kanamycin and geneticin, and the ALS geneencodes resistance to the herbicide chlorsulfuron.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al.,Meth. In Enzymol. 153:253-277 (1987). These vectors are plantintegrating vectors in that on transformation, the vectors integrate aportion of vector DNA into the genome of the host plant. Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene 61:1-11 (1987) and Berger et al., Proc. Natl. Acad.Sci. U.S.A. 86:8402-8406 (1989). Another useful vector herein is plasmidpBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto,Calif.).

A polynucleotide of the present invention can be expressed in eithersense or anti-sense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable plant characteristics. Antisensetechnology can be conveniently used to gene expression in plants. Toaccomplish this, a nucleic acid segment from the desired gene is clonedand operably linked to a promoter such that the anti-sense strand of RNAwill be transcribed. The construct is then transformed into plants andthe antisense strand of RNA is produced. In plant cells, it has beenshown that antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the enzyme of interest, see, e.g.,Sheehy et al., Proc. Nat'l. Acad. Sci. USA 85:8805-8809 (1988); andHiatt et al., U.S. Pat. No. 4,801,340.

Another method of suppression is sense suppression. Introduction ofnucleic acid configured in the sense orientation has been shown to be aneffective means by which to block the transcription of target genes. Foran example of the use of this method to modulate expression ofendogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990) andU.S. Pat. No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res. (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J. Am. Chem. Soc. (1987) 109:1241-1243). Meyer, R.B., et al., J. Am. Chem. Soc. (1989) 111:8517-8519, effect covalentcrosslinking to a target nucleotide using an alkylating agentcomplementary to the single-stranded target nucleotide sequence. Aphotoactivated crosslinking to single-stranded oligonucleotides mediatedby psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988)27:3197-3203. Use of crosslinking in triple-helix forming probes wasalso disclosed by Home, et al., J. Am. Chem. Soc. (1990) 112:2435-2437.Use of N4, N4-ethanocytosine as an alkylating agent to crosslink tosingle-stranded oligonucleotides has also been described by Webb andMatteucci, J. Am. Chem. Soc. (1986) 108:2764-2765; Nucleic Acids Res.(1986) 14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991).Various compounds to bind, detect, label, and/or cleave nucleic acidsare known in the art. See, for example, U.S. Pat. Nos. 5,543,507;5,672,593; 5,484,908; 5,256,648; and, 5,681941.

Proteins

The isolated proteins of the present invention comprise a polypeptidehaving at least 10 amino acids encoded by any one of the polynucleotidesof the present invention as discussed more fully, supra, or polypeptideswhich are conservatively modified variants thereof. Exemplarypolypeptide sequences are provided in SEQ ID NOS: 2, 12, 14, or 22. Theproteins of the present invention or variants thereof can comprise anynumber of contiguous amino acid residues from a polypeptide of thepresent invention, wherein that number is selected from the group ofintegers consisting of from 10 to the number of residues in afull-length cell cycle polypeptide. Optionally, this subsequence ofcontiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acidsin length, often at least 50, 60, 70, 80, or 90 amino acids in length.Further, the number of such subsequences can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includescatalytically active polypeptides of the present invention (i.e.,enzymes). Catalytically active polypeptides have a specific activity atleast 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, andmost preferably at least 80%, 90%, or 95% that of the native(non-synthetic), endogenous polypeptide. Further, the substratespecificity (k_(cat)/K_(m)) is optionally substantially similar to thenative (non-synthetic), endogenous polypeptide. Typically, the K_(m)will be at least 30%, 40%, or 50%, that of the native (non-synthetic),endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or90%. Methods of assaying and quantifying measures of enzymatic activityand substrate specificity (k_(cat)/K_(m)), are well known to those ofskill in the art.

Generally, the proteins of the present invention will, when presented asan immunogen, elicit production of an antibody specifically reactive toa polypeptide of the present invention encoded by a polynucleotide ofthe present invention as described, supra. Exemplary polypeptidesinclude those which are full-length, such as those disclosed in SEQ IDNOS: 2, 12, 14, or 22. Further, the proteins of the present inventionwill not bind to antisera raised against a polypeptide of the presentinvention which has been fully immunosorbed with the same polypeptide.Immunoassays for determining binding are well known to those of skill inthe art. A preferred immunoassay is a competitive immunoassay asdiscussed, infra. Thus, the proteins of the present invention can beemployed as immunogens for constructing antibodies immunoreactive to aprotein of the present invention for such exemplary utilities asimmunoassays or protein purification techniques.

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian, or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so. In eukaryotic cellsoverexpression of a non-functional fusion protein may be desirable.After isolation and purification of the fusion protein from theexpressing cells, enzymatic cleavage could be used to restore functionto the purified CycD protein. In addition, fusions with CycD can haveapplication for affinity matrices and affinity columns used forpurifying other cell cycle genes. For example, “His-patch” thioredoxinfusions can be expressed, and the isolate His-CycD fusion protein boundto metal chelate columns. Whole cell protein extracts can then be passedthrough the column to selectively trap proteins that interact with CycD.See Ausubel et al., 1990 for general methods. Similarly, glutathione-Stransferase fusions can be used to attach proteins to solid-phasematrices for this type of affinity binding. This method has been used,for example, to identify cell cycle genes whose proteins bind to GST-Rbin L. Magnaghi-Jaulin et al., Retinoblastoma protein repressestranscription by recruiting a histone deacetylase. Nature 391:601-604(1998). It may also be advantageous to fuse additional functional genesto the CycD gene. For example it would be useful to fuse a greenfluorescent gene or some other reporter gene.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or inducible) followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither prokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the DNA encoding aprotein of the present invention. To obtain high level expression of acloned gene, it is desirable to construct expression vectors whichcontain, at the minimum, a strong promoter to direct transcription, aribosome binding site for translational initiation, and atranscription/translation terminator. One of skill would recognize thatmodifications can be made to a protein of the present invention withoutdiminishing its biological activity. Some modifications may be made tofacilitate the cloning, expression, or incorporation of the targetingmolecule into a fusion protein. Such modifications are well known tothose of skill in the art and include, for example, a methionine addedat the amino terminus to provide an initiation site, or additional aminoacids (e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purificationsequences.

A. Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambdaderived P L promoter and N-gene ribosome binding site (Shimatake et al.,Nature 292:128 (1981)). The inclusion of selection markers in DNAvectors transfected in E. coli is also useful. Examples of such markersinclude genes specifying resistance to ampicillin, tetracycline, orchloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)).

B. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a of the present invention can beexpressed in these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

Synthesis of heterologous proteins in yeast is well known. Sherman, F.,et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982)is a well recognized work describing the various methods available toproduce the protein in yeast. Suitable vectors usually have expressioncontrol sequences, such as promoters, including 3-phosphoglyceratekinase or other glycolytic enzymes, and an origin of replication,termination sequences and the like as desired. For instance, suitablevectors are described in the literature (Botstein et al., Gene 8:17-24(1979); Broach et al., Gene 8:121-133 (1979)).

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques or radioimmunoassayof other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cell cultures. Mammalian cell systems often will be in theform of monolayers of cells although mammalian cell suspensions may alsobe used. A number of suitable host cell lines capable of expressingintact proteins have been developed in the art, and include the HEK293,BHK21, and CHO cell lines. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. Other animal cells useful for production of proteins of thepresent invention are available, for instance, from the American TypeCulture Collection Catalogue of Cell Lines and Hybridomas (7th edition,1992).

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See Schneider, J.Embryol. Exp. Morphol. 27:353-365 (1987)).

As with yeast, when higher animal or plant host cells are employed,polyadenlyation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenlyation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo, M.,Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA CloningVol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington,Va., pp. 213-238 (1985).

Use in Two-Hybrid Systems

An important utility for the maize CycD genes that have been cloned inthe genetic approach of using a two-hybrid system to identifyinteracting proteins (i.e. proteins that specifically interact with theCycD gene-encoded products. This method, typically done using the yeastSaccharomyces cerevisiae, exploits the fact that a functionaltranscription factor can be separated into two components; a DNA-bindingfactor and an activation domain, which when held together non-covalentlywill still bind DNA and activate transcription. The test system isconstructed as follows: a DNA-binding domain is localized 5′ to areporter gene, for example luciferase, and this cassette is transformedinto a yeast strain. The nucleic acid sequence for the DNA-bindingdomain of the transcriptional factor is ligated to the gene (or partialgene sequence) being used as bait. Expression of this DNA-bindingdomain-bait fusion is driven, for example by the yeast adh1 promoter. A“library” of gene-fusions is also produced, using the activation domainof the transcriptional factor fused to genes (or gene fragments) from anexpression library of interest (referred to as the activation domainhybrid). Expression of the activation domain hybrids is alsoaccomplished, for example, using the yeast adh1 promoter. To perform thetwo-hybrid screen, plasmids encoding the DNA-binding domain hybrid and alibrary of activation domain hybrids are introduced (sequentially orsimultaneously) into a yeast strain already containing the inactivereporter. Transformed yeast in which the activation domain hybridspecifically bind to the DNA-binding domain hybrid will expressluciferase. Positives are further characterized by sequence analysis,and further tests of relevance of biological interactions.

Commonly used DNA-binding domains include those from lexA protein in E.coli, and the Ga14 protein in yeast. Likewise, commonly used activationdomains include B42 (bacterial) and Ga14 (yeast). For details, seeHannon G, and Bartel P, Identification of interacting proteins using thetwo-hybrid system, Methods Mol. Cellular Biol. 5:289-297 (1995).

Transfection/Transformation of Cells

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription and/or translation ofthe sequence to effect phenotypic changes in the organism. Thus, anymethod which provides for efficient transformation/transfection may beemployed.

A. Plant Transformation

A DNA sequence coding for the desired polynucleotide of the presentinvention, for example a cDNA or a genomic sequence encoding a fulllength protein, will be used to construct a recombinant expressioncassette which can be introduced into the desired plant.

Gene Transformation Methods

Numerous methods for introducing foreign genes into plants are known andcan be used to insert the cell cycle gene into a plant host, includingbiological and physical plant transformation protocols. See, forexample, Miki et.al., 1993, “Procedure for Introducing Foreign DNA intoPlants,” In: Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88. Themethods chosen vary with the host plant, and include chemicaltransfection methods such as calcium phosphate, microorganism-mediatedgene transfer such as Agrobacterium (Horsch et al., Science 227:1229-31,1985), electroporation, micro-injection, and biolistic bombardment.

Expression cassettes and vectors and in vitro culture methods for plantcell or tissue transformation and regeneration of plants are known andavailable. See, for example, Gruber et.al., 1993, “Vectors for PlantTransformation” In: Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton,pages 89-119.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants. See, for example,Kado, 1991, Crit. Rev. Plant Sci. 10:1. Descriptions of theAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provide in Gruber et al., supra; Miki et al., supra; andMoloney et al., 1989, Plant Cell Reports 8:238.

Direct Gene Transfer

Methods for Agrobacterium-mediated transformation in rice is disclosedin (Hiei et.al., 1994, The Plant Journal 6:271-282) and maize (Ishida etal., 1996, Nature/Biotechnology 14:745-750). Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation. Methods for Agrobacterium-mediated transformation insorghum are disclosed in WO 98/49332. Methods for Agrobacterium-mediatedtransformation in maize are disclosed in WO 98/32326.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes. (Sanford etal., 1987, Part. Sci. Technol. 5:27; Sanford, 1988, Trends Biotech6:299; Sanford, 1990, Physiol. Plant 79:206; Klein et al., 1992,Biotechnology 10:268).

Another method for physical delivery of DNA to plants is sonication oftarget cells as described in Zang et al., 1991, Bio/Technology 9:996.Alternatively, liposome or spheroplast fusions have been used tointroduce expression vectors into plants. See, for example, Deshayes etal., 1985, EMBO J. 4:2731; and Christou et al., 1987, PNAS USA 84:3962.Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine have also been reported. See, forexample, Hain et al., 1985, Mol. Gen. Genet. 199:161; and Draper et al.,1982, Plant Cell Physiol. 23:451.

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, for example, Donn et al., 1990, In: Abstracts of theVIIth Int'l Congress on Plant Cell and Tissue Culture (IAPTC), A2-38,page 53; D'Halluin et al., 1992, Plant Cell 4:1495-1505; and Spencer etal., 1994, Plant Mol. Biol. 24:51-61. Microinjection of DNA into wholeplant cells has also been described as has microinjection intoprotoplasts. See, for example in whole cells, Neuhaus et al., 1987,Theor. Appl. Genet. 75:30-36; and in protoplasts, Crossway et al., 1986,Mol. Gen. Genet. 202:179-185; and Reich et al., 1986, Biotechnology4:1001-1004.

Particle Wounding/Agrobacterium Delivery

Another useful basic transformation protocol involves a combination ofwounding by particle bombardment, followed by use of Agrobacterium forDNA delivery, as described by Bidney et al., Plant Mol. Biol. 18:301-313(1992). Useful plasmids for plant transformation include PHP9762. Thebinary backbone for PHP9762 is bin 19. See Bevan, Nucleic Acids Research12:8711-8721 (1984).

In general, the intact meristem transformation method involves imbibingseed for 24 hours in the dark, removing the cotyledons and root radical,followed by culturing of the meristem explants. Twenty-four hours later,the primary leaves are removed to expose the apical meristem. Theexplants are placed apical dome side up and bombarded, e.g., twice withparticles, followed by co-cultivation with Agrobacterium. To start theco-cultivation for intact meristems, Agrobacterium is placed on themeristem. After about a 3-day co-cultivation period the meristems aretransferred to culture medium with cefotaxime (plus kanamycin for theNPTII selection). Selection can also be done using kanamycin.

The split meristem method involves imbibing seed, breaking of thecotyledons to produce a clean fracture at the plane of the embryonicaxis, excising the root tip and then bisecting the explantslongitudinally between the primordial leaves. The two halves are placedcut surface up on the medium then bombarded twice with particles,followed by co-cultivation with Agrobacterium. For split meristems,after bombardment, the meristems are placed in an Agrobacteriumsuspension for 30 minutes. They are then removed from the suspensiononto solid culture medium for three day co-cultivation. After thisperiod, the meristems are transferred to fresh medium with cefotaxime(plus kanamycin for selection).

Transfer by Plant Breeding

Once a single transformed plant has been obtained by the foregoingrecombinant DNA method, e.g., a plant transformed with a desired gene,conventional plant breeding methods can be used to transfer thestructural gene and associated regulatory sequences via crossing andbackcrossing. In general, such plant breeding techniques are used totransfer a desired gene into a specific crop plant. In the instantinvention, such methods include the further steps of: (1) sexuallycrossing a transformed plant with a second non-transformed plant; (2)recovering reproductive material from the progeny of the cross; and (3)growing transformed containing plants from the reproductive material.

Isolated nucleic acid acids of the present invention can be introducedinto plants according techniques known in the art. Generally,recombinant expression cassettes as described above and suitable fortransformation of plant cells are prepared. Techniques for transforminga wide variety of higher plant species are well known and described inthe technical, scientific, and patent literature. See, for example,Weising et al., Ann. Rev. Genet. 22:421-477 (1988). For example, the DNAconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation, PEG poration, particlebombardment, silicon fiber delivery, or microinjection of plant cellprotoplasts or embryogenic callus. Alternatively, the DNA constructs maybe combined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria.

The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. 82:5824 (1985). Ballistic transformation techniques aredescribed in Klein et al., Nature 327:70-73 (1987).

Agrobacterium tumefaciens-meditated transformation techniques are welldescribed in the scientific literature. See, for example Horsch et al.,Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci.80:4803 (1983). Although Agrobacterium is useful primarily in dicots,certain monocots can be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, P W J Rigby,Ed., London, Academic Press, 1987; and Lichtenstein, C. P., and Draper,J. In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press,1985), Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, 1984),(3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA87:1228, (1990)).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al., Methods in Enzymology 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlaneMol. Biol. Reporter 6:165 (1988). Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena et al., Nature 325:274 (1987). DNA can alsobe injected directly into the cells of immature embryos and therehydration of desiccated embryos as described by Neuhaus et al., Theor.Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plantviruses that can be employed as vectors are known in the art and includecauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, andtobacco mosaic virus.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,R. J., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977).

Synthesis of Proteins

The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A; Merrifield et al., J. Am. Chem. Soc.85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis,2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greaterlength may be synthesized by condensation of the amino and carboxytermini of shorter fragments. Methods of forming peptide bonds byactivation of a carboxy terminal end (e.g., by the use of the couplingreagent N,N′-dicyclohexylcarbodiimide) is known to those of skill.

Purification of Proteins

The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including selective precipitation with such substances as ammoniumsulfate, column chromatography, immunopurification methods, and others.See, for instance, R. Scopes, Protein Purification: Principles andPractice, Springer-Verlag: New York (1982); Deutscher, Guide to ProteinPurification, Academic Press (1990). For example, antibodies may beraised to the proteins as described herein. Purification from E. colican be achieved following procedures described in U.S. Pat. No.4,511,503. The protein may then be isolated from cells expressing theprotein and further purified by standard protein chemistry techniques asdescribed herein. Detection of the expressed protein is achieved bymethods known in the art and include, for example, radioimmunoassays,Western blotting techniques or immunoprecipitation.

Transgenic Plant Regeneration

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with a polynucleotide of the presentinvention.

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillilan Publishing Company, New York, pp. 124-176 (1983); andBinding, Regeneration of Plants, Plant Protoplasts, CRC Press, BocaRaton, pp. 21-73 (1985).

Transformed plant cells, calli or explant can be cultured onregeneration medium in the dark for several weeks, generally about 1 to3 weeks to allow the somatic embryos to mature. Preferred regenerationmedia include media containing MS salts, such as PHI-E and PHI-F media.The plant cells, calli or explant are then typically cultured on rootingmedium in a light/dark cycle until shoots and roots develop. Methods forplant regeneration are known in the art and preferred methods areprovided by Kamo et al., (Bot. Gaz. 146(3):324-334, 1985), West et al.,(The Plant Cell 5:1361-1369, 1993), and Duncan et al. (Planta165:322-332, 1985).

Small plantlets can then be transferred to tubes containing rootingmedium and allowed to grow and develop more roots for approximatelyanother week. The plants can then be transplanted to soil mixture inpots in the greenhouse.

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaf explants can be achieved as described by Horschet al., Science 227:1229-1231 (1985). In this procedure, transformantsare grown in the presence of a selection agent and in a medium thatinduces the regeneration of shoots in the plant species beingtransformed as described by Fraley et al., Proc. Natl. Acad. Sci. U.S.A.80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987). Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3^(rd) edition, Sprague and Dudley Eds., American Societyof Agronomy, Madison, Wis. (1988).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype, (e.g., altered cell cycle content or composition).

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

Transgenic plants expressing the selectable marker can be screened fortransmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then analyzed for protein expression by Western immunoblot analysisusing the specifically reactive antibodies of the present invention. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered cell division relative to a control plant (i.e., native,non-transgenic). Back-crossing to a parental plant and out-crossing witha non-transgenic plant are also contemplated.

Modulating Cell Cycle Protein Content and/or Composition

The present invention further provides a method for modulating (i.e.,increasing or decreasing) cell cycle protein content or composition in aplant or part thereof. Modulation can be effected by increasing ordecreasing the cell cycle protein content (i.e., the total amount ofcell cycle protein) and/or the cell cycle protein composition (the ratioof various cell cycle monomers in the plant) in a plant. The methodcomprises transforming a plant cell, transiently or stably, with arecombinant expression cassette comprising a polynucleotide of thepresent invention as described above to obtain a transformed plant cell.For stably transformed plant cells, growing the transformed plant cellunder plant forming conditions, and inducing expression of apolynucleotide of the present invention in the plant for a timesufficient to modulate cell cycle protein content and/or composition inthe plant or plant part.

In some embodiments, plant cell division may be modulated by altering,in vivo or in vitro, the promoter of a non-isolated cell cycle gene toup- or down-regulate gene expression. In some embodiments, the codingregions of native cell cycle genes can be altered via substitution,addition, insertion, or deletion to decrease activity of the encodedenzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868. And in some embodiments, an isolated nucleic acid (e.g.,a vector) comprising a promoter sequence is transfected into a plantcell. Subsequently, a plant cell comprising the promoter operably linkedto a polynucleotide of the present invention is selected for by meansknown to those of skill in the art such as, but not limited to, Southernblot, DNA sequencing, or PCR analysis using primers specific to thepromoter and to the gene and detecting amplicons produced therefrom. Aplant or plant part altered or modified by the foregoing embodiments isgrown under plant forming conditions for a time sufficient to modulatecell cycle protein content and/or composition in the plant. Plantforming conditions are well known in the art and discussed briefly,supra.

In general, content or composition is increased or decreased by at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a nativecontrol plant, plant part, or cell lacking the aforementionedrecombinant expression cassette. Modulation in the present invention mayoccur during and/or subsequent to growth of the plant to the desiredstage of development. Modulating nucleic acid expression temporallyand/or in particular tissues can be controlled by employing theappropriate promoter operably linked to a polynucleotide of the presentinvention in, for example, sense or antisense orientation as discussedin greater detail, supra. Induction of expression of a polynucleotide ofthe present invention can also be controlled by exogenous administrationof an effective amount of inducing compound. Inducible promoters andinducing compounds that activate expression from these promoters arewell known in the art. In preferred embodiments, cell division ismodulated in monocots, particularly maize.

Molecular Markers

The present invention provides a method of genotyping a plant comprisinga polynucleotide of the present invention. Genotyping provides a meansof distinguishing homologs of a chromosome pair and can be used todifferentiate segregants in a plant population. Molecular marker methodscan be used for phylogenetic studies, characterizing geneticrelationships among crop varieties, identifying crosses or somatichybrids, localizing chromosomal segments affecting monogenic traits, mapbased cloning, and the study of quantitative inheritance. See, e.g.,Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed.,Springer-Verlag, Berlin (1997). For molecular marker methods, seegenerally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in:Genome Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R.G. Landis Company, Austin, Tex., pp. 7-21.

The particular method of genotyping in the present invention may employany number of molecular marker analytic techniques such as, but notlimited to, restriction fragment length polymorphisms (RFLPs). RFLPs arethe product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. As is well known to those ofskill in the art, RFLPs are typically detected by extraction of genomicDNA and digestion with a restriction enzyme. Generally, the resultingfragments are separated according to size and hybridized with a probe;single copy probes are preferred. Restriction fragments from homologouschromosomes are revealed. Differences in fragment size among allelesrepresent an RFLP. Thus, the present invention further provides a meansto follow segregation of a cell cycle gene or nucleic acid of thepresent invention as well as chromosomal sequences genetically linked tothese genes or nucleic acids using such techniques as RFLP analysis.Linked chromosomal sequences are within 50 centiMorgans (cM), oftenwithin 40 or 30 cM, preferably within 20 or 10 cM, more preferablywithin 5, 3, 2, or 1 cM of a cell cycle gene.

In the present invention, the nucleic acid probes employed for molecularmarker mapping of plant nuclear genomes selectively hybridize, underselective hybridization conditions, to a gene encoding a polynucleotideof the present invention. In preferred embodiments, the probes areselected from polynucleotides of the present invention. Typically, theseprobes are cDNA probes or Pst I genomic clones. The length of the probesis discussed in greater detail, supra, but are typically at least 15bases in length, more preferably at least 20, 25, 30, 35, 40, or 50bases in length. Generally, however, the probes are less than about 1kilobase in length. Preferably, the probes are single copy probes thathybridize to a unique locus in a haploid chromosome complement. Someexemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv,and SstI. As used herein the term “restriction enzyme” includesreference to a composition that recognizes and, alone or in conjunctionwith another composition, cleaves at a specific nucleotide sequence.

The method of detecting an RFLP comprises the steps of (a) digestinggenomic DNA of a plant with a restriction enzyme; (b) hybridizing anucleic acid probe, under selective hybridization conditions, to asequence of a polynucleotide of the present of the genomic DNA; (c)detecting therefrom a RFLP. Other methods of differentiating polymorphic(allelic) variants of polynucleotides of the present invention can behad by utilizing molecular marker techniques well known to those ofskill in the art including such techniques as: 1) single strandedconformation analysis (SSCP); 2) denaturing gradient gel electrophoresis(DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides(ASOs); 5) the use of proteins which recognize nucleotide mismatches,such as the E. coli mutS protein; and 6) allele-specific PCR. Otherapproaches based on the detection of mismatches between the twocomplementary DNA strands include clamped denaturing gel electrophoresis(CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage(CMC). Exemplary polymorphic variants are provided in Table I, supra.Thus, the present invention further provides a method of genotypingcomprising the steps of contacting, under stringent hybridizationconditions, a sample suspected of comprising a polynucleotide of thepresent invention with a nucleic acid probe. Generally, the sample is aplant sample; preferably, a sample suspected of comprising a maizepolynucleotide of the present invention (e.g., gene, mRNA). The nucleicacid probe selectively hybridizes, under stringent conditions, to asubsequence of a polynucleotide of the present invention comprising apolymorphic marker. Selective hybridization of the nucleic acid probe tothe polymorphic marker nucleic acid sequence yields a hybridizationcomplex. Detection of the hybridization complex indicates the presenceof that polymorphic marker in the sample. In preferred embodiments, thenucleic acid probe comprises a polynucleotide of the present invention.

UTR's and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, Nucleic Acids Res. 15:8125(1987)) and the 5<G> 7 methyl GpppG cap structure (Drummond et al.,Nucleic Acids Res. 13:7375 (1985)). Negative elements include stableintramolecular 5′ UTR stem-loop structures (Muesing et al., Cell 48:691(1987)) and AUG sequences or short open reading frames preceded by anappropriate AUG in the 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell.Biol. 8:284 (1988)). Accordingly, the present invention provides 5′and/or 3′ UTR regions for modulation of translation of heterologouscoding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host or tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see Devereaux etal., Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50, or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT publication No.96/19256. See also, Zhang, J. H., et al. Proc. Natl. Acad. Sci. USA94:4504-4509 (1997). Generally, sequence shuffling provides a means forgenerating libraries of polynucleotides having a desired characteristicwhich can be selected or screened for. Libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides which comprise sequence regions which have substantialsequence identity and can be homologously recombined in vitro or invivo. The population of sequence-recombined polynucleotides comprises asubpopulation of polynucleotides which possess desired or advantageouscharacteristics and which can be selected by a suitable selection orscreening method. The characteristics can be any property or attributecapable of being selected for or detected in a screening system, and mayinclude properties of: an encoded protein, a transcriptional element, asequence controlling transcription, RNA processing, RNA stability,chromatin conformation, translation, or other expression property of agene or transgene, a replicative element, a protein-binding element, orthe like, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be anincreased K_(m) and/or K_(cat) over the wild-type protein as providedherein. In other embodiments, a protein or polynculeotide generated fromsequence shuffling will have a ligand binding affinity greater than thenon-shuffled wild-type polynucleotide. The increase in such propertiescan be at least 110%, 120%, 130%, 140% or at least 150% of the wild-typevalue.

Detection of Nucleic Acids

The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of comprising a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of corn. In someembodiments, a cell cycle gene or portion thereof can be amplified priorto the step of contacting the nucleic acid sample with a polynucleotideof the present invention. The nucleic acid sample is contacted with thepolynucleotide to form a hybridization complex. The polynucleotidehybridizes under stringent conditions to a gene encoding a polypeptideof the present invention. Formation of the hybridization complex is usedto detect a gene encoding a polypeptide of the present invention in thenucleic acid sample. Those of skill will appreciate that an isolatednucleic acid comprising a polynucleotide of the present invention shouldlack cross-hybridizing sequences in common with non-cell cycle genesthat would yield a false positive result.

Detection of the hybridization complex can be achieved using any numberof well-known methods. For example, the nucleic acid sample, or aportion thereof, may be assayed by hybridization formats including butnot limited to, solution phase, solid phase, mixed phase, or in situhybridization assays. Briefly, in solution (or liquid) phasehybridizations, both the target nucleic acid and the probe or primer arefree to interact in the reaction mixture. In solid phase hybridizationassays, probes or primers are typically linked to a solid support wherethey are available for hybridization with target nucleic in solution. Inmixed phase, nucleic acid intermediates in solution hybridize to targetnucleic acids in solution as well as to a nucleic acid linked to a solidsupport. In in situ hybridization, the target nucleic acid is liberatedfrom its cellular surroundings in such as to be available forhybridization within the cell while preserving the cellular morphologyfor subsequent interpretation and analysis. The following articlesprovide an overview of the various hybridization assay formats: Singeret al., Biotechniques 4(3):230-250 (1986); Haase et al., Methods inVirology, Vol. VII, pp. 189-226 (1984); Wilkinson, The theory andpractice of in situ hybridization in: In situ Hybridization, D. G.Wilkinson, Ed., IRL Press, Oxford University Press, Oxford; and NucleicAcid Hybridization: A Practical Approach, Hames, B. D. and Higgins, S.J., Eds., IRL Press (1987).

Nucleic Acid Labels and Detection Methods

The means by which nucleic acids of the present invention are labeled isnot a critical aspect of the present invention and can be accomplishedby any number of methods currently known or later developed. Detectablelabels suitable for use in the present invention include any compositiondetectable by spectroscopic, radioisotopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include biotin for staining with labeledstreptavidin conjugate, magnetic beads, fluorescent dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

Nucleic acids of the present invention can be labeled by any one ofseveral methods typically used to detect the presence of hybridizednucleic acids. One common method of detection is the use ofautoradiography using probes labeled with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P, orthe like. The choice of radio-active isotope depends on researchpreferences due to ease of synthesis, stability, and half lives of theselected isotopes. Other labels include ligands which bind to antibodieslabeled with fluorophores, chemiluminescent agents, and enzymes.Alternatively, probes can be conjugated directly with labels such asfluorophores, chemiluminescent agents or enzymes. The choice of labeldepends on sensitivity required, ease of conjugation with the probe,stability requirements, and available instrumentation. Labeling thenucleic acids of the present invention is readily achieved such as bythe use of labeled PCR primers.

In some embodiments, the label is simultaneously incorporated during theamplification step in the preparation of the nucleic acids. Thus, forexample, polymerase chain reaction (PCR) with labeled primers or labelednucleotides will provide a labeled amplification product. In anotherembodiment, transcription amplification using a labeled nucleotide(e.g., fluorescein-labeled UTP and/or CTP) incorporates a label into thetranscribed nucleic acids.

Non-radioactive probes are often labeled by indirect means. For example,a ligand molecule is covalently bound to the probe. The ligand thenbinds to an anti-ligand molecule that is either inherently detectable orcovalently bound to a detectable signal system, such as an enzyme, afluorophore, or a chemiluminescent compound. Enzymes of interest aslabels will primarily be hydrolases, such as phosphatases, esterases andglycosidases, or oxidoreductases, particularly peroxidases. Fluorescentcompounds include fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, etc. Chemiluminescers includeluciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. Ligands andanti-ligands may be varied widely. Where a ligand has a naturalanti-ligand, namely ligands such as biotin, thyroxine, and cortisol, itcan be used in conjunction with its labeled, naturally-occurringanti-ligands. Alternatively, any haptenic or antigenic compound can beused in combination with an antibody.

Probes can also be labeled by direct conjugation with a label. Forexample, cloned DNA probes have been coupled directly to horseradishperoxidase or alkaline phosphatase, (Renz. M., and Kurz, K., AColorimetric Method for DNA Hybridization, Nucl. Acids Res. 12:3435-3444(1984)) and synthetic oligonucleotides have been coupled directly withalkaline phosphatase (Jablonski, E., et al., Preparation ofOligodeoxynucleotide-Alkaline Phosphatase Conjugates and Their Use asHybridization Probes, Nuc. Acids. Res. 14:6115-6128 (1986); and Li P.,et al., Enzyme-linked Synthetic Oligonucleotide probes: Non-RadioactiveDetection of Enterotoxigenic Escherichia Coli in Faeca Specimens, Nucl.Acids Res. 15:5275-5287 (1987)).

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

Antibodies to Proteins

Antibodies can be raised to a protein of the present invention,including individual, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated. Personsof skill know many methods of making antibodies. The followingdiscussion is presented as a general overview of the techniquesavailable; however, one of skill will recognize that many variationsupon the following methods are known.

A number of immunogens are used to produce antibodies specificallyreactive with a protein of the present invention. An isolatedrecombinant, synthetic, or native cell cycle protein of 5 amino acids inlength or greater and selected from a protein encoded by apolynucleotide of the present invention are the preferred immunogens(antigen) for the production of monoclonal or polyclonal antibodies.Those of skill will readily understand that the proteins of the presentinvention are typically denatured, and optionally reduced, prior toformation of antibodies for screening expression libraries or otherassays in which a putative protein of the present invention is expressedor denatured in a non-native secondary, tertiary, or quaternarystructure. Naturally occurring cell cycle polypeptides can be usedeither in pure or impure form.

The protein of the present invention is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated for subsequent use in immunoassays tomeasure the presence and quantity of the protein of the presentinvention. Methods of producing polyclonal antibodies are known to thoseof skill in the art. In brief, an immunogen (antigen), preferably apurified protein, a protein coupled to an appropriate carrier (e.g.,GST, keyhole limpet hemanocyanin, etc.), or a protein incorporated intoan immunization vector such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the protein of interest. When appropriately high titersof antibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the protein is performed wheredesired (See, e.g., Coligan, Current Protocols in Immunology,Wiley/Greene, NY (1991); and Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Press, NY (1989)).

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of a protein of thepresent invention are raised by immunizing animals, e.g., withconjugates of the fragments with carrier proteins as described above.Typically, the immunogen of interest is a protein of at least about 5amino acids, more typically the protein is 10 amino acids in length,preferably, 15 amino acids in length and more preferably the protein is20 amino acids in length or greater. The peptides are typically coupledto a carrier protein (e.g., as a fusion protein), or are recombinantlyexpressed in an immunization vector. Antigenic determinants on peptidesto which antibodies bind are typically 3 to 10 amino acids in length.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. Monoclonals antibodies are screened for binding to a proteinfrom which the immunogen was derived. Specific monoclonal and polyclonalantibodies will usually have an antibody binding site with an affinityconstant for its cognate monovalent antigen at least between 10⁶-10⁷,usually at least 10⁸, preferably at least 10⁹, more preferably at least10¹⁰, and most preferably at least 10¹¹ liters/mole.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Basic and Clinical Immunology, 4th ed., Stites et al.,Eds., Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies:Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986);and Kohler and Milstein, Nature 256:495-497 (1975). Summarized briefly,this method proceeds by injecting an animal with an immunogen comprisinga protein of the present invention. The animal is then sacrificed andcells taken from its spleen, which are fused with myeloma cells. Theresult is a hybrid cell or “hybridoma” that is capable of reproducing invitro. The population of hybridomas is then screened to isolateindividual clones, each of which secrete a single antibody species tothe immunogen. In this manner, the individual antibody species obtainedare the products of immortalized and cloned single B cells from theimmune animal generated in response to a specific site recognized on theimmunogenic substance.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse et al., Science246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989); andVaughan et al., Nature Biotechnology, 14:309-314 (1996)). Alternatively,high avidity human monoclonal antibodies can be obtained from transgenicmice comprising fragments of the unrearranged human heavy and lightchain Ig loci (i.e., minilocus transgenic mice). Fishwild et al., NatureBiotech., 14:845-851 (1996). Also, recombinant immunoglobulins may beproduced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al., Proc.Nat'l Acad. Sci. 86:10029-10033 (1989).

The antibodies of this invention are also used for affinitychromatography in isolating proteins of the present invention. Columnsare prepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified protein arereleased.

The antibodies can be used to screen expression libraries for particularexpression products such as normal or abnormal protein. Usually theantibodies in such a procedure are labeled with a moiety allowing easydetection of presence of antigen by antibody binding.

Antibodies raised against a protein of the present invention can also beused to raise anti-idiotypic antibodies. These are useful for detectingor diagnosing various pathological conditions related to the presence ofthe respective antigens.

Frequently, the proteins and antibodies of the present invention will belabeled by joining, either covalently or non-covalently, a substancethat provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

Protein Immunoassays

Means of detecting the proteins of the present invention are notcritical aspects of the present invention. In a preferred embodiment,the proteins are detected and/or quantified using any of a number ofwell-recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology, Vol. 37:Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds.(1991). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, e.g., those reviewed inEnzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980);Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniquesin Biochemistry and Molecular Biology, Elsevier Science Publishers B.V.,Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A PracticalGuide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles andPractice of Immunoassays, Price and Newman Eds., Stockton Press, NY(1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY(1988). Immunological binding assays (or immunoassays) typically utilizea “capture agent” to specifically bind to and often immobilize theanalyte (in this case, a protein of the present invention). The captureagent is a moiety that specifically binds to the analyte. In a preferredembodiment, the capture agent is an antibody that specifically binds aprotein(s) of the present invention. The antibody may be produced by anyof a number of means known to those of skill in the art as describedherein.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledprotein of the present invention or a labeled antibody specificallyreactive to a protein of the present invention. Alternatively, thelabeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/protein complex.

In a preferred embodiment, the labeling agent is a second antibodybearing a label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond can be modified with a detectable moiety, such as biotin, towhich a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (See,generally Kronval et al., J. Immunol. 111: 401-1406 (1973), andAkerstrom et al., J. Immunol. 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

While the details of the immunoassays of the present invention may varywith the particular format employed, the method of detecting a proteinof the present invention in a biological sample generally comprises thesteps of contacting the biological sample with an antibody whichspecifically reacts, under immunologically reactive conditions, to aprotein of the present invention. The antibody is allowed to bind to theprotein under immunologically reactive conditions, and the presence ofthe bound antibody is detected directly or indirectly.

A. Non-Competitive Assay Formats

Immunoassays for detecting proteins of the present invention includecompetitive and noncompetitive formats. Noncompetitive immunoassays areassays in which the amount of captured analyte (i.e., a protein of thepresent invention) is directly measured. In one preferred “sandwich”assay, for example, the capture agent (e.g., an antibody specificallyreactive, under immunoreactive conditions, to a protein of the presentinvention) can be bound directly to a solid substrate where they areimmobilized. These immobilized antibodies then capture the proteinpresent in the test sample. The protein thus immobilized is then boundby a labeling agent, such as a second antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second can bemodified with a detectable moiety, such as biotin, to which a thirdlabeled molecule can specifically bind, such as enzyme-labeledstreptavidin.

B. Competitive Assay Formats

In competitive assays, the amount of analyte present in the sample ismeasured indirectly by measuring the amount of an added (exogenous)analyte (e.g., a protein of the present invention) displaced (orcompeted away) from a capture agent (e.g., an antibody specificallyreactive, under immunoreactive conditions, to the protein) by theanalyte present in the sample. In one competitive assay, a known amountof analyte is added to the sample and the sample is then contacted witha capture agent that specifically binds a protein of the presentinvention. The amount of protein bound to the capture agent is inverselyproportional to the concentration of analyte present in the sample.

In a particularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of protein bound to the antibody may bedetermined either by measuring the amount of protein present in aprotein/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of protein may be detected byproviding a labeled protein.

A hapten inhibition assay is another preferred competitive assay. Inthis assay a known analyte, (such as a protein of the present invention)is immobilized on a solid substrate. A known amount of antibodyspecifically reactive, under immunoreactive conditions, to the proteinis added to the sample, and the sample is then contacted with theimmobilized protein. In this case, the amount of antibody bound to theimmobilized protein is inversely proportional to the amount of proteinpresent in the sample. Again, the amount of immobilized antibody may bedetected by detecting either the immobilized fraction of antibody or thefraction of the antibody that remains in solution. Detection may bedirect where the antibody is labeled or indirect by the subsequentaddition of a labeled moiety that specifically binds to the antibody asdescribed above.

C. Generation of Pooled Antisera for Use in Immunoassays

A protein that specifically binds to or that is specificallyimmunoreactive with an antibody generated against a defined immunogen,such as an immunogen consisting of the amino acid sequence of SEQ IDNOS: 2, 12, 14, or 22, is determined in an immunoassay. The immunoassayuses a polyclonal antiserum which is raised to a polypeptide of thepresent invention (i.e., the immunogenic polypeptide). This antiserum isselected to have low crossreactivity against other proteins and any suchcrossreactivity is removed by immunoabsorbtion prior to use in theimmunoassay (e.g., by immunosorbtion of the antisera with a protein ofdifferent substrate specificity (e.g., a different enzyme) and/or aprotein with the same substrate specificity but of a different form).

In order to produce antisera for use in an immunoassay, a polypeptide isisolated as described herein. For example, recombinant protein can beproduced in a mammalian or other eukaryotic cell line. An inbred strainof mice is immunized with the protein of using a standard adjuvant, suchas Freund's adjuvant, and a standard mouse immunization protocol (seeHarlow and Lane, supra). Alternatively, a synthetic polypeptide derivedfrom the sequences disclosed herein and conjugated to a carrier proteinis used as an immunogen. Polyclonal sera are collected and titeredagainst the immunogenic polypeptide in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁴ or greater are selectedand tested for their cross reactivity against polypeptides of differentforms or substrate specificity, using a competitive binding immunoassaysuch as the one described in Harlow and Lane, supra, at pages 570-573.Preferably, two or more distinct forms of polypeptides are used in thisdetermination. These distinct types of polypeptides are used ascompetitors to identify antibodies that are specifically bound by thepolypeptide being assayed for. The competitive polypeptides can beproduced as recombinant proteins and isolated using standard molecularbiology and protein chemistry techniques as described herein.

Immunoassays in the competitive binding format are used forcrossreactivity determinations. For example, the immunogenic polypeptideis immobilized to a solid support. Proteins added to the assay competewith the binding of the antisera to the immobilized antigen. The abilityof the above proteins to compete with the binding of the antisera to theimmobilized protein is compared to the immunogenic polypeptide. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith a distinct form of a polypeptide are selected and pooled. Thecross-reacting antibodies are then removed from the pooled antisera byimmunoabsorbtion with a distinct form of a polypeptide.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described herein to compare a second “target”polypeptide to the immunogenic polypeptide. In order to make thiscomparison, the two polypeptides are each assayed at a wide range ofconcentrations and the amount of each polypeptide required to inhibit50% of the binding of the antisera to the immobilized protein isdetermined using standard techniques. If the amount of the targetpolypeptide required is less than twice the amount of the immunogenicpolypeptide that is required, then the target polypeptide is said tospecifically bind to an antibody generated to the immunogenic protein.As a final determination of specificity, the pooled antisera is fullyimmunosorbed with the immunogenic polypeptide until no binding to thepolypeptide used in the immunosorbtion is detectable. The fullyimmunosorbed antisera is then tested for reactivity with the testpolypeptide. If no reactivity is observed, then the test polypeptide isspecifically bound by the antisera elicited by the immunogenic protein.

D. Other Assay Formats

In a particularly preferred embodiment, Western blot (immunoblot)analysis is used to detect and quantify the presence of protein of thepresent invention in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind a protein of the present invention. Theantibodies specifically bind to the protein on the solid support. Theseantibodies may be directly labeled or alternatively may be subsequentlydetected using labeled antibodies (e.g., labeled sheep anti-mouseantibodies) that specifically bind to the antibodies.

E. Quantification of Proteins

The proteins of the present invention may be detected and quantified byany of a number of means well known to those of skill in the art. Theseinclude analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

F. Reduction of Non-Specific Binding

One of skill will appreciate that it is often desirable to reducenon-specific binding in immunoassays and during analyte purification.Where the assay involves an antigen, antibody, or other capture agentimmobilized on a solid substrate, it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this involves coating the substrate with a proteinaceouscomposition. In particular, protein compositions such as bovine serumalbumin (BSA), nonfat powdered milk, and gelatin are widely used.

G. Immunoassay Labels

The labeling agent can be, e.g., a monoclonal antibody, a polyclonalantibody, a binding protein or complex, or a polymer such as an affinitymatrix, carbohydrate or lipid. Detectable labels suitable for use in thepresent invention include any composition detectable by spectroscopic,radioisotopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Detection may proceed by any known method,such as immunoblotting, western analysis, gel-mobility shift assays,fluorescent in situ hybridization analysis (FISH), tracking ofradioactive or bioluminescent markers, nuclear magnetic resonance,electron paramagnetic resonance, stopped-flow spectroscopy, columnchromatography, capillary electrophoresis, or other methods which tracka molecule based upon an alteration in size and/or charge. Theparticular label or detectable group used in the assay is not a criticalaspect of the invention. The detectable group can be any material havinga detectable physical or chemical property. Such detectable labels havebeen well-developed in the field of immunoassays and, in general, anylabel useful in such methods can be applied to the present invention.Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads, fluorescent dyes, radiolabels, enzymes, and colorimetric labelsor colored glass or plastic beads, as discussed for nucleic acid labels,supra.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, ease of conjugation of thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, e.g., by microscopy,visual inspection, via photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple colorimetric labels may be detectedsimply by observing the color associated with the label. Thus, invarious dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Assays for Compounds that Modulate Enzymatic Activity or Expression

The present invention also provides means for identifying compounds thatbind to (e.g., substrates), and/or increase or decrease (i.e., modulate)the activity of active polypeptides of the present invention. The methodcomprises contacting a polypeptide of the present invention with acompound whose ability to bind to or modulate enzyme activity is to bedetermined. The polypeptide employed will have at least 20%, preferablyat least 30% or 40%, more preferably at least 50% or 60%, and mostpreferably at least 70% or 80% of the specific activity of the native,full-length cell cycle polypeptide (e.g., enzyme). Generally, thepolypeptide will be present in a range sufficient to determine theeffect of the compound, typically about 1 nM to 10 μM. Likewise, thecompound will be present in a concentration of from about 1 nM to 10 μM.Those of skill will understand that such factors as enzymeconcentration, ligand concentrations (i.e., substrates, products,inhibitors, activators), pH, ionic strength, and temperature will becontrolled so as to obtain useful kinetic data and determine thepresence of absence of a compound that binds or modulates polypeptideactivity. Methods of measuring enzyme kinetics are well known in theart. See, e.g., Segel, Biochemical Calculations, 2^(nd) ed., John Wileyand Sons, New York (1976).

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

Clones of ZmCycDa-1 and ZmCycDc-1 are on deposit with the American TypeCulture Collection (ATCC). The ATCC is at 10801 University Boulevard,Manassas, Va. 20110-2209. The deposits have been made under the terms ofthe Budapest Treaty and given the ATCC designation 98848 and 98847respectively.

During the pendency of this patent application, access to the depositedcultures will be available to the Commissioner of Patents and Trademarksand to persons determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and 35 U.S.C. 122.

All restrictions imposed by the depositor on the availability to thepublic of the deposited material will be irrevocably removed upon thegranting of a patent. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

EXAMPLES Example 1 Isolation of Maize CycD Genes

Total RNA was isolated from corn tissues with TRIzol Reagent (LifeTechnology Inc. Gaithersburg, Md.) using a modification of the guanidineisothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi[Chomczynski, P., and Sacchi, N., Anal. Biochem. 162, 156 (1987)]. Inbrief, plant tissue samples were pulverized in liquid nitrogen beforethe addition of the TRIzol Reagent, and then were further homogenizedwith a mortar and pestle. Addition of chloroform followed bycentrifugation was conducted for separation of an aqueous phase and anorganic phase. The total RNA was recovered by precipitation withisopropyl alcohol from the aqueous phase.

Poly(A)+ RNA Isolation:

The selection of poly(A)+ RNA from total RNA was performed usingPolyATract system (Promega Corporation. Madison, Wis.). In brief,biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed using high stringency conditions and eluted using RNase-freedeionized water.

cDNA Library Construction:

cDNA synthesis was performed and unidirectional cDNA libraries wereconstructed using the SuperScript Plasmid System (Life Technology Inc.Gaithersburg, Md.). The first stand of cDNA was synthesized by primingan oligo(dT) primer containing a Not I site. The reaction was catalyzedby SuperScript Reverse Transcriptase II at 45° C. The second strand ofcDNA was labeled with alpha-³²P-dCTP and a portion of the reaction wasanalyzed by agarose gel electrophoresis to determine cDNA sizes. cDNAmolecules smaller than 500 base pairs and unligated adapters wereremoved by Sephacryl-S400 chromatography. The selected cDNA moleculeswere ligated into pSPORT1 vector in between Not I and Sal I sites.Mitotically active tissues from Zea mays were employed, including suchsources as shoot cultures, immature inflorescences (tassel and ear) aswell as other sources of vegetative meristems.

Sequencing Template Preparation:

Individual colonies were picked and DNA was prepared either by PCR withM13 forward primers and M13 reverse primers, or by plasmid isolation.All the cDNA clones were initially sequenced using M13 reverse primers.As additional fragments of the genes were discovered, new sequencingprimers were designed.

PROTOCOLS, Murray (ed.), pages 271-281 (Humana Press, Inc. 1991).Functional fragments of the cell cycle protein are identified by theirability, upon introduction to cells, to stimulate the G1 to S-phasetransition, which is manifested by increased DNA replication in apopulation of cells and by increased cell division rates.

5′-RACE

Library RACE was performed using several of Pioneer's maize libraries.5′ RACE was done using a cDNA library constructed from leaves and stemsof maize plants at the three-leaf stage. The principal of 5′ RACE isdescribed in detail in numerous publications such as: Frohman M. A.1993. Rapid Amplification of Complementary DNA Ends for Generation ofFull-Length Complementary DNAs: Thermal RACE. In: Methods in Enzymology,vol. 28, pp 340-356. Detailed procedure can be found in the ClonTechMarathon cloning manual.

Example 2 Using CycD's in a Two-Hybrid System to Identify Maize CellCycle Genes

CycD gene expression during the G1→S transition and early S-phase play aprominent role in progression through the cell cycle. The proteinsencoded by the CycD gene family are a critical part of the complex thatbinds and phosphorylates retinoblastoma-associated gene family members.In turn, Rb releases E2F and this transcription factor starts thecascade of events leading to DNA replication. As such, the CycD genesand their encoded proteins can be used to identify other cell cycleregulatory proteins. This can be done using the CycD gene as bait (thetarget fused to the DNA-binding domain) in a yeast two-hybrid screen.Methods for two-hybrid library construction, cloning of the reportergene, cloning of the DNA-binding and activation domain hybrid genecassettes, yeast culture, and transformation of the yeast are all doneaccording to well-established methods (see Sambrook et al., 1990;Ausubel et al., 1990; Hannon and Bartels, 1995). Using this method, Zeamays Cdc2, Cdk4 and Rb genes are identified as components of theactivation domain hybrid, and are confirmed through further sequenceanalysis. Similarly, inhibitors of the Cdk4/CycD complex such as CIP andInk are identified.

Example 3 CycD-Bound Affinity Columns for Identifying Cdk4 Proteins andtheir Encoding Genes

Purified recombinant CycD protein can be immobilized on a matrix via acovalent crosslinking or affinity purification as described supra. Thismatrix can then be used to pull-down proteins that interact with CycDproteins, inter alia, cyclin-dependent kinase. CDK activity can then beassessed by measuring the addition of radioactive phosphorus toprotein-substrates and CDK protein levels determined by immunoassay.Additionally, this can be used to purify the CDK activity present indifferent plant tissues and protein fractions. The presence and level ofother CycD interacting proteins can also be determined on the basis ofimmunological assay, activity quantification, SDS-PAGE analysis andother methods. These measures can then be correlated with thereproductive state, capacity for division, developmental stage, or thequality of different samples. A CycD nucleic acid can also be adductedto a second nucleic acid sequence encoding a DNA-binding domain in orderto identify CycD interacting proteins.

Example 4 Transient CycD Expression Stimulates DNA Replication andEnhances Transgene Integration

Regardless of the method of DNA delivery, cells competent for theintegration of foreign DNA must be actively dividing. There is a growingbody of evidence suggesting that integration of foreign DNA occurs individing cells (this includes both Agrobacterium and direct DNA deliverymethods). It has long been observed that dividing transformed cellsrepresent only a fraction of cells that transiently express a transgene.It is well known (in non-plant systems) that the delivery of damagedDNA, (similar to what we introduce by particle gun delivery methods)induces an immediate cell cycle arrest, a process involving cyclindependent kinase inhibitors (CDKI's). This inhibition can be obviated byectopic transient over-expression of positive cell cycle regulators orby down-regulation of negative regulators. Regardless of the mechanismof arrest; i.e. presence of damaged DNA or delivery into a non-cyclingdifferentiated cell, stimulation of the cell cycle will increaseintegration frequencies. To demonstrate this, the CycD gene is clonedinto a cassette with a constitutive promoter (i.e. either a strong maizepromoter such as the ubiquitin promoter including the first ubiquitinintron, or a weak constitutive promoter such as nos). Delivery of theZmCycD gene in an appropriate plant expression cassette (for example, ina UBI::ZmCycD::pinII-containing plasmid) along with UBI::bar::pinII canbe accomplished through numerous well-established methods for plantcells, including for example particle bombardment, sonication, PEGtreatment or electroporation of protoplasts, electroporation of intacttissue, silica-fiber methods, microinjection or Agrobacterium-mediatedtransformation. Using one of the above methods, DNA is introduced intomaize cells capable of growth on suitable maize culture medium. Suchcompetent cells can be from maize suspension culture, callus culture onsolid medium, freshly isolated immature embryos or meristem cells.Immature embryos of the Hi-II genotype are used as the target forco-delivery of these two plasmids. Transient expression of the CycD geneovercomes the G1/S checkpoint controls, and increases the proportion ofrecipient-cells (i.e. into which DNA was introduced) that enter S-phase.This stimulation through the G1/S transition in cells harboringtransgenic plasmid DNA provides an optimal cellular environment forintegration of the introduced genes. Cytological methods can be used toverify increased frequencies of progression through S-phase and mitosis(i.e. for cells in which a visual marker such as GFP was transformedalongside CycD the green fluorescent cells will exhibit a higher mitoticindex). Cells in S-phase (undergoing DNA replication) can be monitoredby detecting nucleotide analog incorporation. For example, followingincubation of cells with bromodeoxyuridine (BrdU) incorporation of thisthymadine analog can be detected by methods such as antiBrdUimmunocytochemistry or through enhancement of Topro3 fluorescencefollowing BrdU labeling. It is expected that CycD expression willincrease the proportion of cells incorporating BrdU (i.e. a higherpercentage of transformed cells will incorporate BrdU relative tountransformed cells). Increased DNA synthesis can also be monitoredusing such methods as fluorescence activated cell sorting (FACS) ofprotoplasts (or nuclei), in conjunction with appropriateBrdU-insensitive fluorescent DNA labels such as propidium iodide andDAPI or BrdU-detecting methods described above. For example, tissue ishomogenized to release nuclei that are analyzed using the FACS for bothgreen fluorescence (from our accompanying GFP marker) and DNA content.Such FACS analysis can demonstrate that expression of a co-transformedGFP reporter correlates with CycD-induced changes in the ratios of cellsin G1, S and G2. Similar experiments can be run using the fluorescentlylabeled anti-BrdU antisera to demonstrate that CycD expression increasedthe percentage of cells in S-phase. Cell cycle stage-specific probes canalso be used to monitor cell cycle progression. For example, numerousspindle-associated proteins are expressed during a fairly narrow windowduring mitosis, and antibodies or nucleic acid probes to cyclins,histones, or DNA synthesis enzymes can be used as positive markers forthe G1/S transition. For cells that have received the CycD genecassette, stimulation of the cell cycle is manifested in an increasedmitotic index, detected by staining for mitotic figures using a DNA dyesuch as DAPI or Hoechst 33258. FACS analysis of CycD-expressing cells isexpected to show that a high percentage of cells have progressed into orthrough S-phase. Progression through S-phase will be manifested by fewercells in G1 and/or more rapid cycling times (i.e. shorter G1 and G2stages). A higher percentage of cells are labeled when cell cyclestage-specific probes are used, as mentioned above.

To assess the effect on transgene integration, growth ofbialaphos-resistant colonies on selective medium is a reliable assay.Within 1-7 days after DNA introduction, the embryos are moved ontoculture medium containing 3 mg/I of the selective agent bialaphos.Embryos, and later callus, are transferred to fresh selection platesevery 2 weeks. After 6-8 weeks, transformed calli are recovered.Transgenic callus containing the introduced genes can be verified usingPCR and Southern analysis. Northern analysis can also be used to verifywhich calli are expressing the bar gene, and whether the CycD gene isbeing expressed at levels above normal wild-type cells (based onhybridization of probes to freshly isolated mRNA population from thecells). In immature embryos that had transient, elevated CycDexpression, higher numbers of stable transformants are recovered (likelya direct result of increased integration frequencies). Increasedtransgene integration frequency can also be assessed using suchwell-established labeling methods such as in situ hybridization.

For this specific application (using transient CycD-mediated cell cyclestimulation to increase transient integration frequencies), it may bedesirable to reduce the likelihood of ectopic stable expression of theCycD gene. Strategies for transient-only expression can be used. Thisincludes delivery of RNA (transcribed from the CycD gene) or CycDprotein along with the transgene cassettes to be integrated to enhancetransgene integration by transient stimulation of cell division. Usingwell-established methods to produce CycD-RNA, this can then be purifiedand introduced into maize cells using physical methods such asmicroinjection, bombardment, electroporation or silica fiber methods.For protein delivery, the gene is first expressed in a bacterial orbaculoviral system, the protein purified and then introduced into maizecells using physical methods such as microinjection, bombardment,electroporation or silica fiber methods. Alternatively, CycD proteinsare delivered from Agrobacterium tumefaciens into plant cells in theform of fusions to Agrobacterium virulence proteins. Fusions areconstructed between CycD and bacterial virulence proteins such as VirE2,VirD2, or VirF which are known to be delivered directly into plantcells. Fusions are constructed to retain both those properties ofbacterial virulence proteins required to mediate delivery into plantcells and the CycD activity required for enhancing transgeneintegration. This method should ensure a high frequency of simultaneousco-delivery of T-DNA and functional CycD protein into the same hostcell. The methods above represent various means of using the CycD geneor its encoded product to transiently stimulate DNA replication and celldivision, which in turn enhances transgene integration by providing animproved cellular/molecular environment for this event to occur.

Example 5 Altering CycD Expression Stimulated the Cell Cycle, IncreasingIntegration and Growth

Based on results in other eukaryotes, expression of ZmCycD genesstimulates the G1/S transition and promotes cell division. This increasein division rate is assessed in a number of different manners, morerapid incorporation of radiolabeled nucleotides, and faster growth (i.e.more biomass accumulation). Delivery of the ZmCycD in an appropriateplant expression cassette is accomplished through numerouswell-established methods for plant cells, including for example particlebombardment, sonication, PEG treatment or electroporation ofprotoplasts, electroporation of intact tissue, silica-fiber methods,microinjection or Agrobacterium-mediated transformation. The result ofZmCycD gene expression will be to stimulate the G1/S transition andhence cell division, providing the optimal cellular environment forintegration of introduced genes (as per Example 1). This will trigger atissue culture response (cell divisions) in genotypes that typically donot respond to conventional culture techniques, or stimulate growth oftransgenic tissue beyond the normal rates observed in wild-type(non-transgenic) tissues. To demonstrate this, the CycD gene (ZmCycDc-1)was cloned into a cassette with a constitutive promoter (the ubiquitinpromoter, UBI, including the first ubiquitin intron). Particlebombardment was used to introduce the UBI::ZmCycDc-1::pinII-containingplasmid along with a UBI::PAT˜GFP::pinII-containing plasmid (which, whenexpressed produced a functional PAT˜GFP fusion protein which conferredbialaphos resistance and green fluorescence) into maize cells capable ofgrowth on suitable maize culture medium. Such competent cells can befrom maize suspension culture, callus culture on solid medium, freshlyisolated immature embryos or meristem cells. Immature embryos of theHi-II genotype were used as the target for co-delivery of these twoplasmids. Ears were harvested at approximately 10 days post-pollination,and 1.2-1.5 mm immature embryos were isolated from the kernels. Theimmature embryos were bombarded from 18-72 hours later. Typically, theimmature embryos were placed on a high-osmoticum medium for 6-18 hoursprior to bombardment, and were left on this medium for an additional 18hours after bombardment.

DNA Particle Bombardment

Between 6 and 18 hours prior to bombardment, the immature embryos wereplaced on medium with additional osmoticum (MS basal medium, Musashigeand Skoog, 1962, Physiol. Plant 15:473-497, with 0.25 M sorbitol). Theembryos on the high-osmotic medium were used as the bombardment target.

For particle bombardment, plasmid DNA (described above) was precipitatedonto 1.8 μm tungsten particles using standard CaCl₂-spermidine chemistry(see, for example, Klein et al., 1987, Nature 327:70-73). Each plate wasbombarded once at 600 PSI, using a DuPont Helium Gun (Lowe et al., 1995,Bio/Technol 13:677-682). For typical media formulations used for maizeimmature embryo isolation, callus initiation, callus proliferation andregeneration of plants, see Armstrong, C. 1994. In “The Maize Handbook”,M. Freeling and V. Walbot, eds. Springer Verlag, NY, pp 663-671.

Selection

Within 1-7 days the embryos were moved onto N6-based culture mediumcontaining 3 mg/l of the selective agent bialaphos. Embryos, and latercallus, were transferred to fresh selection plates every 2 weeks. Afterthe first 14 days post-bombardment, the calli developing from theimmature embryos were screened for GFP expression using anepifluorescent dissecting-microscope. Typically, (i.e. in the absence ofa cell cycle gene) this is too early to observe growing multicellulartransformants. Instead, as typical after such a short post-bombardmentduration, numerous GFP-expressing single-cells were observed on controlembryos (where the UBI::PAT˜GFP::pinII plasmid was introduced alone),but GFP-expressing multicellular clusters were not observed. In markedcontrast to the control treatment, when UBI::CycDc-1 was included alongwith the PAT˜GFP marker, numerous GFP+multicellular clusters wereobserved growing from the immature embryos at this same earlytime-point. This early stimulation and higher number of growingtransformants observed in the CycD treatment, suggest that expression ofthis cell cycle gene increased integration frequencies (thus highernumbers) and stimulated growth of these small colonies after integrationhad occurred (thus, the transformants were clearly visible at this earlyjuncture). After 6-8 weeks, transformed calli were recovered. Intreatments where both the PAT˜GFP gene and CycD were transformed intoimmature embryos, a higher number of growing calli were recovered on theselective medium and callus growth was stimulated (relative totreatments with the bar gene alone). In the first comparativeexperiments of this type, immature embryos were harvested from 30 ears(over a period of 3 months). From each ear, 25 embryos were used for thecontrol and 25 embryos were used for the UBI::CycD treatment. Thus thetotal number of embryos used per treatment was 750. The transformationfrequency (the number of transgene-expressing independent calli relativeto the starting number of embryos) for the control treatment was 2.4%.For the UBI::CycDc-1 treated embryos, the transformation frequency hadincreased to 7.2%.

A second experiment demonstrated that both the maize CycDa-1 and CycDc-1genes result in increased transformation frequencies relative to thecontrol treatment (where the cyclin gene was not included). For thisbombardment experiment (performed in a similar manner to that describedabove), 3 Hi-II ears were harvested at 10 DAP, and the immature embryoswere divided evenly between the 3 treatments (125 embryos pertreatment). Again, transformants appeared at earlier timepoints in thetwo CycD treatments and the final number of transformants in the CycDtreatments was substantially higher. When screened for GFP expression 46days post-bombardment, no GFP-expressing multicellular calli wereobserved in the control treatment, while in the CycDc-1 and CycDa-1treatments there were macroscopic GFP+ calli at frequencies of 0.7 and2.3%, respectively. After 77 days, the overall transformation frequencyfor the control was 7.4%, while for CycDc-1 and CycDa-1 the frequencyhad increased to 12.0 and 18.3% respectively. In addition, the calli inthe CycD treatments were substantially larger than in the controltreatment, indicating that these genes stimulated growth rates.

Differences in cell cycle profiles were also observed in CycD-expressingcells relative to control (wild-type) cells. To demonstrate thatoverexpression of CycD genes could accelerate cell division, the cellcycle profile of maize calli expressing Ubi::CycD were analyzed using acell sorter (flow cytometry assay). Flow cytometry is a standard methodto study cell cycle, using procedures that are well established in theliterature, as, for example, in Sonea I M et al., Am J Vet Res. 199960(3):346-53. Briefly, by counting the number of cells that are in G1phase versus the number of cells that are in G2 phase, one can estimate,in a given population, the percentage of cells that are undergoing celldivision. The higher the percentage of cells in G1 phase, the less thenumber of cells that are dividing. Under standard culture conditions,approximately 70% of the G1/G2 cells of maize calli are in the G1 phase.In maize calli expressing CycD genes, alterations of the distribution ofcells in the G1 and G2 phases were observed. In 14 out of 19 CycDa-1expressing events, the proportion of cells in G1 phase decreased tobelow 60%, and in some cases dropped below 30%. Thus, in these 14CycDa-1 events, more cells were undergoing cell division compared towild type maize calli. Using a different CycD gene also altered the cellcycle of transformants, but not in as many events. Compared to the 14out of 19 CycDa-1 expressing events with increased cell division rates,only two out of 32 CycDc-1 expressing events showed that the percentageof G1 cells was lower than 60%. In control calli expressing similarvector genes but lacking a CycD gene, the cell cycle profile remainedsimilar to that of the non-treated wild type maize calli.

Calli from both the CycDa-1 and CycDc-1 treatment regenerated easily.Healthy, fertile transgenic plants were grown in the greenhouse.Seed-set on CycD transgenic plants was similar to control plants, andtransgenic progeny were recovered.

For a given CycD gene, it was also observed that higher expressionlevels improved transformation. For this bombardment experiment(performed in a similar manner to that described above), 3 Hi-II earswere harvested at 10 DAP, and the immature embryos were divided evenlybetween the 3 treatments (125 embryos per treatment). The treatmentsincluded a no-cyclin control (UBI::PAT˜GFP::pinII), or theUBI::PAT˜GFP::pinII marker plus one of three cyclin-expressing plasmids(UBI::CycDc-1, nos::CycDc-1 or UBI::Da-1). For the CycDc-1 gene, thisexperiment compared high levels of cyclin expression (UBI) to low levels(nos). The transformation frequency in the control treatment was 3.0%.When expression was driven by the UBI promoter, the transformationfrequencies for the CycDa-1 and CycDc-1 genes were 14.4 and 17.6%,respectively. However, placing the CycDc-1 gene behind the nos promoterresulted in a transformation similar to the control (1.6%). Based onthis result, it appears that higher expression levels result incorrespondingly higher recovery of transformants.

Example 6 Identifying Transformants in the Absence of Chemical Selection

When the CycD gene is introduced without any additional selectivemarker, transgenic calli can be identified by their ability to grow morerapidly than surrounding wild-type (non-transformed) tissues. Transgeniccallus can be verified using PCR and Southern analysis. Northernanalysis can also be used to verify which calli are expressing the bargene, and which are expressing the maize CycD gene at levels abovenormal wild-type cells (based on hybridization of probes to freshlyisolated mRNA population from the cells).

Inducible Expression:

The CycD gene can also be cloned into a cassette with an induciblepromoter such as the benzenesulfonamide-inducible promoter. Theexpression vector is co-introduced into plant cells and after selectionon bialaphos, the transformed cells are exposed to the safener(inducer). This chemical induction of CycD expression should result instimulated G1/S transition and more rapid cell division. The cells arescreened for the presence of ZmCycD RNA by northern, or RT-PCR (usingtransgene specific probes/oligo pairs), for CycD-encoded protein usingCycD-specific antibodies in Westerns or using hybridization. IncreasedDNA replication is detected using BrdU labeling followed by antibodydetection of cells that incorporated this thymidine analogue. Likewise,other cell cycle division assays could be employed, as described above.

Example 7 Control of CycD Gene Expression Using Tissue-Specific orCell-Specific Promoters Provides a Differential Growth Advantage

CycD gene expression using tissue-specific or cell-specific promotersstimulates cell cycle progression in the expressing tissues or cells.For example, using a seed-specific promoter will stimulate cell divisionrate and result in increased seed biomass. Alternatively, driving CycDexpression with a strongly-expressed, early, tassel-specific promoterwill enhance development of this entire reproductive structure.

Expression of CycD genes in other cell types and/or at different stagesof development will similarly stimulate cell division rates. Similar toresults observed in Arabidopsis (Doerner et al., 1996), root-specific orroot-preferred expression of CycD will result in larger roots and fastergrowth (i.e. more biomass accumulation).

Example 8 Meristem Transformation

Meristem transformation protocols rely on the transformation of apicalinitials or cells that can become apical initials followingreorganization due to injury or selective pressure. The progenitors ofthese apical initials differentiate to form the tissues and organs ofthe mature plant (i.e. leaves, stems, ears, tassels, etc.). Themeristems of most angiosperms are layered with each layer having its ownset of initials. Normally in the shoot apex these layers rarely mix. Inmaize the outer layer of the apical meristem, the L1, differentiates toform the epidermis while descendants of cells in the inner layer, theL2, give rise to internal plant parts including the gametes. Theinitials in each of these layers are defined solely by position and canbe replaced by adjacent cells if they are killed or compromised.Meristem transformation frequently targets a subset of the population ofapical initials and the resulting plants are chimeric. If for example, 1of 4 initials in the L1 layer of the meristem are transformed only ¼ ofepidermis would be transformed. Selective pressure can be used toenlarge sectors but this selection must be non-lethal since large groupsof cells are required for meristem function and survival. Transformationof an apical initial with a Cyclin D expression cassette under theexpression of a promoter active in the apical meristem (either meristemspecific or constitutive) would allow the transformed cells to growfaster and displace wildtype initials driving the meristem towardshomogeneity and minimizing the chimeric nature of the plant body. Todemonstrate this, the CycD gene is cloned into a cassette with apromoter that is active within the meristem (i.e. either a strongconstitutive maize promoter such as the ubiquitin promoter including thefirst ubiquitin intron, or a promoter active in meristematic cells suchas the maize histone, cdc2 or actin promoter). Coleoptilar stage embryosare isolated and plated meristem up on a high sucrose maturation medium(see Lowe et al., 1997). The cyclin D expression cassette along with areporter construct such as Ubi:GUS:pinII can then be co-delivered(preferably 24 hours after isolation) into the exposed apical dome usingconventional particle gun transformation protocols. As a control theCycD construct can be replaced with an equivalent amount of pUC plasmidDNA. After a week to 10 days of culture on maturation medium the embryoscan be transferred to a low sucrose hormone-free germination medium.Leaves from developing plants can be sacrificed for GUS staining.Transient expression of the CycD gene in meristem cells, throughstimulation of the G1→S transition, will result in greater integrationfrequencies and hence more numerous transgenic sectors. Integration andexpression of the CycD gene will impart a competitive advantage toexpressing cells resulting in a progressive enlargement of thetransgenic sector. Due to the enhanced growth rate in CycD-expressingmeristem cells, they will supplant wild-type meristem cells as the plantcontinues to grow. The result will be both enlargement of transgenicsectors within a given cell layer (i.e. periclinal expansion) and intoadjacent cell layers (i.e. anticlinal invasions). As an increasinglylarge proportion of the meristem is occupied by CycD-expressing cells,the frequency of CycD germline inheritance should go up accordingly.

Example 9 Use of Flp/Frt System to Excise the CycD Cassette

In cases where the CycD gene has been integrated and CycD expression isuseful in the recovery of maize trangenics, but is ultimately notdesired in the final product, the CycD expression cassette (or anyportion thereof that is flanked by appropriate FRT recombinationsequences) can be excised using FLP-mediated recombination (see U.S.patent application Ser. No. 08/972,258 filed Nov. 18, 1997).

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

1. A method for increasing transformation frequency of a maize cellcomprising introducing into the maize cell an isolated cyclin D (CycD)polynucleotide operably linked to a promoter; wherein said CycDpolynucleotide encodes a polypeptide comprising at least 95% sequenceidentity to SEQ ID NO: 22 and wherein the % identity is determined byGCG/bestfit program using a gap creation penalty of 50 and a gapextension penalty of 3; wherein when the CycD polypeptide is expressedfrom said CycD polynucleotide, said maize cell has increasedtransformation efficiency when compared to a maize cell without anintroduced isolated CycD polynucleotide; and wherein said maize CycDpolypeptide binds to cyclin-dependent kinase 4 (CDK4)
 2. The method ofclaim 1, wherein said CycD polynucleotide encodes SEQ ID NO:
 22. 3. Themethod of claim 1, wherein said CycD polynucleotide is SEQ ID NO:
 21. 4.An isolated nucleic acid encoding a polypeptide which increasestransformation frequency in a maize cell comprising: (a) apolynucleotide encoding a polypeptide comprising at least 95% sequenceidentity to SEQ ID NO: 22, wherein the % identity is determined byGCG/bestfit program using a gap creation penalty of 50 and a gapextension penalty of 3; (b) a polynucleotide encoding SEQ ID NO: 22; (c)a polynucleotide of SEQ ID NO: 21; or (d) a polynucleotide fullycomplementary to a polynucleotide of (a), (b), or (c).
 5. A recombinantexpression cassette comprising the nucleic acid of claim 4 operablylinked to a promoter.
 6. A host cell comprising the nucleic acid ofclaim 4 stably incorporated in its genome.
 7. The host cell of claim 6,wherein the host cell is a plant cell.
 8. The host cell of claim 7,wherein the plant cell is from a plant selected from the groupconsisting of corn, soybean, sorghum, sunflower, safflower, wheat, rice,alfalfa, or Brassica.
 9. The host cell of claim 6, wherein the host cellis in a plant.
 10. The host cell of claim 9, wherein the plant isselected from the group consisting of corn, soybean, sorghum, sunflower,safflower, wheat, rice, alfalfa, or Brassica.
 11. The host cell of claim6, wherein the host cell is in a seed.
 12. The host cell of claim 11,wherein the seed is from a plant selected from the group consisting ofcorn, soybean, sorghum, sunflower, safflower, wheat, rice, alfalfa, orBrassica.